AU2023201682A1

AU2023201682A1 - Protein proximity assay in formalin fixed paffafin embedded tissue using caged haptens

Info

Publication number: AU2023201682A1
Application number: AU2023201682A
Authority: AU
Inventors: Yuri Belosludtsev; Traci D. Degeer; Wendy J. French; Junshan HAO; Brian Daniel Kelly; Adrian MURILLO; Nathan Walter Polaske
Original assignee: Ventana Medical Systems Inc
Current assignee: Ventana Medical Systems Inc
Priority date: 2015-08-28
Filing date: 2023-03-17
Publication date: 2023-04-13
Anticipated expiration: 2036-08-26
Also published as: JP2018528425A; US20220089622A1; US11053266B2; JP2019532275A; CN116338167A; CN109791144B; AU2021218128B2; AU2021218128A1; CA2996798A1; JP6880001B2; US20180186821A1; CN109791144A; AU2016316846B2; US20190233447A1; US11345719B2; AU2016316846A1; WO2018039605A1; JP7033122B2; AU2023201682B2; EP3341734A4

Abstract

PROTEIN PROXIMITY ASSAY IN FORMALIN FIXED PAFF AFIN EMBEDDED TISSUE USING CAGED HAPTENS Abstract IDd 101 0104 103A 10A o I J.a 10 102 101 102 101 102 106 107 rflO:N~g106 1)04 W035 N~ 9 0 10U1 1402 101 102 FIG.2 Disclosed herein are caged haptens and caged hapten-antibody conjugates useful for enabling the detection of targets located proximally to each other in a sample.

Description

P296830D2 1

PROTEIN PROXIMITY ASSAY IN FORMALIN FIXED PAFFAFIN EMBEDDED TISSUE USING CAGED HAPTENS CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62/301,489 filed February 29, 2016, and the benefit of the filing date of U.S. Provisional Patent Application No. 62/211,590 filed August 28, 2015, the disclosures of which are hereby incorporated herein by reference in their entireties. The present application is a divisional of AU 2021218128, which itself is a divisional of AU 2016316846, which is the national phase entry of PCT/US2016/049153, the entire disclosures of which are incorporated herein by cross-reference.

FIELD OF THE INVENTION

Disclosed embodiments concern detecting targets in a sample, including targets located proximally in a sample. Disclosed embodiments also provide for a proximity assay for detecting protein dimers in formalin-fixed, paraffin embedded tissue using caged haptens or caged hapten conjugates.

STATEMENT OF INDUSTRIAL APPLICABILITY

The present disclosure has industrial applicability in the fields of chemistry and diagnostics.

BACKGROUND OF THE INVENTION

Immunohistochemistry (IHC)refers to the processes of detecting, localizing, and/or quantifying antigens, such as a protein, in a biological sample using antibodies specific to the particular antigens. IHC provides the substantial advantage of identifying exactly where a particular protein is located within the tissue sample. It is also an effective way to examine the tissues themselves. In situ hybridization (ISH) refers to the process of detecting, localizing, and quantifying nucleic acids. Both IHC and ISH can be performed on various biological samples, such as tissue (e.g. fresh frozen, formalin fixed, paraffin embedded) and cytological samples. Recognition of the targets can be detected using various labels (e.g., chromogenic, fluorescent, luminescent, radiometric), irrespective of whether the target is a nucleic acid or an antigen. To robustly detect, locate, and quantify targets in a clinical setting, amplification of the recognition event is desirable as the ability to confidently detect cellular markers of low abundance becomes increasingly important for diagnostic purposes. For example, depositing at the marker's site hundreds or thousands of label molecules in response to a single antigen detection event enhances, through amplification, the ability to detect that recognition event.

Networks of protein-protein interactions are the hallmarks of biological

systems. Protein-protein interactions form signal pathways that regulate all aspects

of cellular functions in normal and cancerous cells. Methods have been developed

for detecting protein-protein interactions, such as transient receptor tyrosine kinase

dimerization and complex formation after extracellular growth factor activation;

however, these methods are not particularly designed to be used on formalin fixed

paraffin embedded (FFPE) tissues. The ability to interrogate for presence and distribution of specific

intermolecular interactions for biomarkers known to be important determinants in

cancer biology is of high interest in the context of new diagnostic capabilities and

for determining therapeutic effect in the context of pharmaceutical development.

The ability to probe and document distributions of molecular interactions on frozen

and paraffin embedded tissue has remained inaccessible; alternative technologies

to approach this question have been proposed, although the solutions have not

proven to be effective and reliable under practical use.

A proximity ligation assay has recently been developed by OLink AB.

This is the only known commercial product for in situ detection of protein-protein

interactions on formalin fixed paraffin embedded tissue. Proximity ligation assay

technology uses DNA ligases to generate a padlock circular DNA template, as well

as Phi29 DNA polymerase for rolling circle amplification. These enzymes are

expensive. Moreover, these enzymes are not amenable for use with automated

systems and methods. For these reasons, proximity ligation assays are not

considered generally useful for commercial applications.

BRIEF SUMMARY OF THE INVENTION Applicants have developed a superior method of detecting targets located

proximally in a sample. In one aspect of the present disclosure is a method for

analyzing a sample to determine whether a first target is proximal to a second target, the method comprising: (a) forming a second target-unmasking enzyme antibody conjugate complex; (b) forming a first target-caged hapten-antibody conjugate complex; (c) unmasking the caged hapten of the first target-caged hapten-antibody conjugate complex to form a first target-unmasked hapten antibody conjugate complex; (d) contacting the sample with first detection reagents to label the first target-unmasked hapten-antibody conjugate complex or the first target; and (e) detecting the labeled first target-unmasked hapten-antibody conjugate complex or labeled first target.

In some embodiments, the first detection reagents comprise (i) a secondary

antibody specific to the unmasked hapten of the first target-unmasked hapten

antibody complex, the secondary antibody conjugated to a first enzyme such that

the secondary antibody labels the first target-unmasked hapten-antibody complex

with the first enzyme; and (ii) a first substrate for the first enzyme. In some

embodiments, the first substrate is a chromogenic substrate or a fluorescent

substrate. In some embodiments, the first detection reagents include amplification

components to label the unmasked enzyme of the first target-unmasked hapten

antibody conjugate complex with a plurality of first reporter moieties. In some

embodiments, the plurality of first reporter moieties are haptens. In some

embodiments, the first detection reagents further comprise secondary antibodies

specific to the plurality of first reporter moieties, each secondary antibody

conjugated to a second reporter moiety. In some embodiments, the second reporter

moiety is selected from the group consisting of an amplification enzyme or a

fluorophore. In some embodiments, the second reporter moiety is an amplification

enzyme and wherein the first detection reagents further comprise a first

chromogenic substrate or a fluorescent substrate for the amplification enzyme. In

some embodiments, the method further comprises contacting the sample with a

second substrate specific for the unmasking enzyme of the second target

unmasking enzyme-antibody conjugate complex and detecting signals

corresponding to a product of a reaction between the second substrate and the

unmasking enzyme.

In another aspect of the present disclosure is a method for analyzing a

sample to determine whether a first target is proximal to a second target, the

method comprising: (a) forming a second target-unmasking enzyme-antibody conjugate complex; (b) forming a first target-caged hapten-antibody conjugate complex; (c) unmasking the caged hapten of the first target-caged hapten-antibody conjugate complex to form a first target-unmasked hapten-antibody conjugate complex; (d) performing a signal amplification step to label the first target unmasked hapten-antibody conjugate complex with a plurality of reporter moieties; and (e) detecting the plurality of reporter moieties.

In some embodiments, the plurality of reporter moieties are haptens; and

wherein the method further comprises introducing secondary antibodies specific to

the plurality of first reporter moieties, each secondary antibody conjugated to a

second reporter moiety. In some embodiments, the second reporter moiety is an

amplification enzyme and wherein the method further comprises introducing a

some embodiments, the method further comprises detecting a total amount of

target in the sample.

In another aspect of the present disclosure is a method for analyzing a

sample to determine whether a first target is proximal to a second target, the

method comprising: (a) forming a second target-unmasking enzyme-antibody

conjugate complex; (b) forming a first target-caged hapten-antibody conjugate

complex; (c) performing a decaging step such that an unmasking enzyme of the

second target-unmasking enzyme-antibody conjugate complex reacts with an

enzyme substrate portion of the first target-caged hapten-antibody conjugate

complex to form a first target-unmasked hapten-antibody conjugate complex; (d)

contacting the sample with first detection reagents to label the first target

unmasked hapten-antibody conjugate complex or the first target; and (e) detecting

the labeled first target-unmasked hapten-antibody conjugate complex or labeled

first target.

In some embodiments, the decaging step comprises changing the

temperature of the sample. In some embodiments, the decaging step comprises

altering a pH of the sample. In some embodiments, the decaging step comprises

introducing one or more washing steps. In some embodiments, the decaging step

comprises adding cofactors for the unmasking enzyme. In some embodiments, the

method further comprises detecting a total amount of target in the sample.

In another aspect of the present disclosure is a method for analyzing a

sample to determine whether a first target is proximal to a second target, the

method comprising: (a) contacting the sample with a caged hapten-antibody

conjugate specific to the first targettoformafirsttarget-caged hapten-antibody

conjugate complex; (b) contacting the sample with an unmasking enzyme-antibody

conjugate specific to the second target to form a second target-unmasking enzyme

antibody conjugate complex, wherein an unmasking enzyme of the unmasking

enzyme-antibody conjugate is selected such that it is capable of reacting with an

enzyme substrate portion of the caged hapten-antibody conjugate to form a first

target-unmasked hapten-antibody conjugate complex; (c) contacting the sample

with first detection reagents to label the first target-unmasked hapten-antibody

conjugate complex or the first target; and (d) detecting the labeled first target

unmasked hapten-antibody conjugate complex or labeled first target.

In some embodiments, a caged hapten portion of the caged hapten-antibody

conjugate is derived from a hapten selected from the group consisting of DCC,

biotin, nitropyrazole, thiazolesulfonamide, benzofurazan, and 2

hydroxyquinoxaline. In some embodiments, the unmasking enzyme of the

unmasking enzyme-antibody conjugate is selected from the group consisting of

alkaline phosphatase, B-glucosidase, B-Galactosidase, B-Glucuronidase, Lipase,

Sulfatase, Amidase, Protease, Nitroreductase, beta-lactamase, neuraminidase, and

Urease. In some embodiments, the first detection reagents comprise (i) a secondary

antibody specific to the unmasked hapten of the first target-unmasked hapten

antibody complex, the secondary antibody conjugated to a first enzyme such that

the secondary antibody labels first target-unmasked hapten-antibody complex with

the first enzyme; and (ii) a first chromogenic substrate or fluorescent substrate. In

some embodiments, the first enzyme is different than the unmasking enzyme. In

some embodiments, the first enzyme is a peroxidase. In some embodiments, the

first chromogenic substrate is selected from the group consisting of 3,3'

diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), HRP-Silver, and tyramide-chromogens. In some embodiments, the method further comprises

contacting the sample with a second chromogenic substrate or fluorescent substrate

specific for the unmasking enzyme of the second target-unmasking enzyme

antibody conjugate complex, wherein the first and second chromogenic substrates are different. In some embodiments, the first detection reagents include components to amplify the amount of label introduced to the first target-unmasked hapten-antibody conjugate complex. In some embodiments, the first target is one of PD-1 or PD-Li, and the second target is the other of PD-1 or PD-L.

In another aspect of the present disclosure is a method for analyzing a

sample to determine whether a first target is proximal to a second target, the

method comprising: (a) contacting the sample with a first detection probe, the first

detection probe comprising one of a caged hapten-antibody conjugate or an

unmasking enzyme-antibody conjugate; (b) contacting the sample with a second

detection probe, the second detection probe comprising the other of the caged

hapten-antibody conjugate or the unmasking enzyme-antibody conjugate; (c)

contacting the sample with at least first detection reagents to label a formed

unmasked hapten-antibody conjugate target complex; (d) detecting signals from

the labeled unmasked hapten-antibody conjugate target complex.

In some embodiments, the method further comprises detecting a total

amount of target within the sample. In some embodiments, the first detection

reagents include amplification components to label the unmasked enzyme of the

first target-unmasked hapten-antibody conjugate complex with a plurality of first

reporter moieties. In some embodiments, the plurality of first reporter moieties are

haptens. In some embodiments, the first detection reagents further comprise

secondary antibodies specific to the plurality of first reporter moieties, each

secondary antibody conjugated to a second reporter moiety. In some embodiments,

the second reporter moiety is selected from the group consisting of an

amplification enzyme or a fluorophore. In some embodiments, the second reporter

moiety is an amplification enzyme and wherein the first detection reagents further

comprise a first chromogenic substrate or fluorescent substrate for the

amplification enzyme. In some embodiments, the method further comprises a

decaging step.

In another aspect of the present disclosure is a method for analyzing a

sample to determine whether a first target is proximal to a second target, the

method comprising: (a) contacting the sample with a caged hapten-antibody

conjugate specific to the first targettoformafirsttarget-caged hapten-antibody

conjugate complex; (b) contacting the sample with an unmasking enzyme-antibody conjugate specific to the second target to form a second target-unmasking enzyme antibody conjugate complex, wherein an unmasking enzyme of the unmasking enzyme-antibody conjugate is selected such that it is capable of reacting with an enzyme substrate portion of the caged hapten-antibody conjugate to form a first target-unmasked hapten-antibody conjugate complex; (c) contacting the sample with a first labeling conjugate that specifically binds to the first target-unmasked hapten-antibody conjugate complex, wherein the first labeling conjugate comprises a first enzyme which differs from the unmasking enzyme; (d) contacting the sample with a first signaling conjugate comprising a first latent reactive moiety and a first chromogenic or fluorescent moiety; and (e) detecting signals from the first chromogenic or fluorescent moiety, the first signals being indicative of proximal first and second targets.

In some embodiments, the method further comprises contacting the sample

with a second signaling conjugate comprising a second latent reactive moiety and a

second chromogenic or fluorescent moiety, wherein the first and second

chromogenic or fluorescent moieties provide different signals. In some

embodiments, further comprises detecting signals from the second chromogenic

moiety, the signals from the second chromogenic or fluorescent moiety being

indicative of total protein.

In another aspect of the present disclosure is a caged hapten having Formula

(I):

Y X-Hapten A- -Caging Group q (I), wherein

Y is selected from a carbonyl-reactive group, an amine-reactive group, or a

thiol-reactive group;

X is a bond, or a group comprising a branched or unbranched, substituted

or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 30

carbon atoms, and optionally having one or more heteroatoms selected from the

group consisting of 0, N, or S;

A is a bond, or a group comprising a branched or unbranched, substituted

or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15

carbon atoms, and optionally having one or more heteroatoms selected from the

group consisting of 0, N, or S; and

'Caging Group' comprises an enzyme substrate and optionally a leaving

group, wherein the leaving group portion comprises a substituted or unsubstituted

5-, 6-, or 7-membered aromatic or heterocyclic ring.

In some embodiments, the 5-, 6-, or 7-membered aromatic or heterocyclic

ring is substituted with a moiety selected from the group consisting of a halogen, a

-S-alkyl group having between 1 and 4 carbon atoms; an -0-alkyl group having

between 1 and 4 carbon atoms; a -N(H)-alkyl group having between 1 and 4

carbon atoms; a -N-(alkyl) 2 group having between 1 and 6 carbon atoms; and a

branched or unbranched, substituted or unsubstituted alkyl group having between 1

and 4 carbon atoms. In some embodiments, the caging group has the structure of

Formula (II):

R2

R1 R1

wherein

R is independently selected from H, F, Cl, Br, I, -0-methyl, -0--ethyl, O-n-propyl, -0--iso-propyl; -0-n-butyl, -0-sec-butyl, or -0-iso-butyl; a -S alkyl group having between 1 and 4 carbon atoms; an -0-alkyl group having

between 1 and 4 carbon atoms; a -N(H)-alkyl group having between 1 and 4

carbon atoms; a -N-(alkyl) 2 group having between 1 and 6 carbon atoms; an alkyl

group having between 1 and 4 carbon atoms and optionally substituted with N or

S; cyano groups; and carboxyl groups; and

R2 is an enzyme substrate.

In some embodiments, the enzyme substrate is selected from the group

consisting of a phosphate group, an ester group, an amide group, a sulfate group, a glycoside group, a urea group, and a nitro group. In some embodiments, the caging group has the structure of Formula (IB):

R2

R1 R(

and

wherein R2 is selected from the group consisting of a phosphate group, an

ester group, an amide group, a sulfate group, a glycoside group, a urea group, and a

nitro group. In some embodiments, each Rg roup is different.

The caged hapten of claim 43, wherein the caging group has the structure

of Formula (IIC):

Ri

(IC), and

wherein R2 is selected from the group consisting of a phosphate group, an

nitro group. In some embodiments, each Rg roup is different.

In some embodiments, the caging group has the structure of Formula (IID):

R2

R1 R1

(IID)), and

wherein R2 is selected from the group consisting of a phosphate group, an ester group, an amide group, a sulfate group, a glycoside group, a urea group, and a nitro group. In some embodiments, each R group is different. In some embodiments, X has the structure of Formula (IIIA):

Ra

Q - e (IIIA),

wherein d and e are integers each independently ranging from 4 to 18; Q is a bond, 0, S, or N(R')(Rd); Ra and R are independently H, a C1 -C 4 alkyl group, F, Cl, or N(R )(Rd); and R' and Rd are independently CH 3 or H. In some embodiments, d and e are integers each independently ranging from 1 to 24. In some embodiments, the caged hapten is selected from the group consisting of:

0 - OH HO Et H

0 HO ff 0 P <Ho NH OH 0 T H:

00

HO-P-OH H,,

0 0 C02

NO0 - NN N 02- N cIP"

011 HO-P -OH HO 0

OO

N 0

H iH/0

HO-P -OH 0

O\ /OH0 N OOH 0 N 0 0 0 N ' N N

HH

a HOH

H HO 0H

0

HO-P -OH 0

'-N

N 0

0 H

0

0 0 aN N

and 0

In another aspect of the present disclosure is a conjugate of (i) a specific

binding entity, and (ii) a caged hapten, including any of the caged haptens cited

herein. In some embodiments, the caged hapten has the structure of Formula (I):

Y -X -Hapten A Caging Group -frlq (I) wherein

Y is selected from a carbonyl-reactive group, an amine-reactive group, or a

thiol-reactive group;

X is a bond, or a group comprising a branched or unbranched, substituted

carbon atoms, and optionally having one or more heteroatoms selected from the

group consisting of 0, N, or S;

A is a bond, or a group comprising a branched or unbranched, substituted

carbon atoms, and optionally having one or more heteroatoms selected from the

group consisting of 0, N, or S; and

'Caging Group' comprises an enzyme substrate and optionally a leaving

5-, 6-, or 7-membered aromatic or heterocyclic ring.

In some embodiments, the specific binding entity of the conjugate is an

antibody. In some embodiments, the conjugate has the structure of Formula (IV):

Antibody Z- Hapten A-]-Caging Group] q n (IV), wherein Z is a bond, or a group comprising a branched or unbranched,

substituted or unsubstituted, saturated or unsaturated aliphatic group having

between 1 and 30 carbon atoms, and optionally having one or more heteroatoms

selected from the group consisting of 0, N, or S,

A is a bond, or a group comprising a branched or unbranched, substituted

or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of 0, N, or S; qisOor 1; and n is an integer ranging from 1 to 25.

In some embodiments, the caging group is a 5-, 6-, or 7-membered

aromatic or heterocyclic ring optionally substituted with a moiety selected from the

group consisting of a halogen, a -S-alkyl group having between 1 and 4 carbon

atoms; an -0-alkyl group having between 1 and 4 carbon atoms; a -N(H)-alkyl

group having between 1 and 4 carbon atoms; a -N-(alkyl) 2 group having between 1

and 6 carbon atoms; and a branched or unbranched, substituted or unsubstituted

alkyl group having between 1 and 4 carbon atoms.

In some embodiments, the caging group has the structure of Formula (II):

R2

R1 R1

wherein

R 1 is independently selected from H, F, Cl, Br, I, -0-methyl, -0--ethyl,

O-n-propyl, -0--iso-propyl; -0-n-butyl, -0-sec-butyl, or -0-iso-butyl; a -S alkyl group having between 1 and 4 carbon atoms; an -0-alkyl group having

between 1 and 4 carbon atoms; a -N(H)-alkyl group having between 1 and 4

group having between 1 and 4 carbon atoms and optionally substituted with N or

S; cyano groups; and carboxyl groups; and

R2 is an enzyme substrate.

In some embodiments, the enzyme substrate is selected from the group

consisting of a phosphate group, an ester group, an amide group, a sulfate group, a

glycoside group, a urea group, and a nitro group.

In some embodiments, the caging group has the structure of Formula (IB):

R2

R1R

(IIB), and

wherein R2 is selected from the group consisting of a phosphate group, an

nitro group. In some embodiments, each Rg roup is different.

In some embodiments, the caging group has the structure of Formula (IIC):

Ri

R2 R1

(1IC), and

wherein R 2 is selected from the group consisting of a phosphate group, an

nitro group. In some embodiments, Rg roup is different.

In some embodiments, the caging group has the structure of Formula (IID):

R2

R1 R1

(IID)), and

wherein R2 is selected from the group consisting of a phosphate group, an

nitro group. In some embodiments, each Rg roup is different.

In some embodiments, the antibody is a secondary antibody. In some

embodiments, the antibody is a primary antibody. In some embodiments, the

primary antibody is specific for a target selected from the group consisting of PD

L1/CD80(B7-1); CTLA-4/CD80(B7-1); CTLA-4/CD86(B7-1); PD-L2/PD-1; any combination of a ErbB family (Herl (EGFR), Her2, Her3, Her4); any combination

of DNA mixed match repair proteins (MLH1, MLH3, MSH2, MSH3, MSH6, PMS1 and PMS2); post translational modifications (PTM) - phosphorylated proteins (combining an anti-phosphotyrosine / phosphoserine / phosphothreonine /

phosphohistidine antibody with any antibody specific against the target in

question); and PTM - ubiquitinated proteins.

In another aspect of the present disclosure is a method for detecting

multiple targets within a sample comprising: (a) contacting the sample with a

caged hapten-antibody conjugate specific to the first target to form a first target

caged hapten-antibody conjugate complex; (b) contacting the sample with an

unmasking enzyme-antibody conjugate specific to the second target to form a

second target-unmasking enzyme-antibody conjugate complex, wherein an

unmasking enzyme of the unmasking enzyme-antibody conjugate is selected such

that it is capable of reacting with an enzyme substrate portion of the caged hapten

antibody conjugate to form a first target-unmasked hapten-antibody conjugate

complex; (c) contacting the sample with first detection reagents to label the first target-unmasked hapten-antibody conjugate complex or the first target; (d) contacting the sample with a first detection probe specific to a third target to form a third target-detection probe complex; (e) contacting the sample with second detection reagents to label the third target-detection probe complex; (f) detecting the labeled first target-unmasked hapten-antibody conjugate complex or labeled first target; and (g) detecting the labeled third target-detection probe complex. In some embodiments, the method further comprises detecting total protein within the sample. In some embodiments, the first detection probe comprises an antibody. In some embodiments, the first detection probe comprises a nucleic acid probe. In some embodiments, the method further comprised contacting the sample with a second detection probe specific to a fourth target to form a fourth target-detection probe complex. In some embodiments, the method further comprises inactivating the unmasking enzyme prior to contacting the sample with second detection reagents. In some embodiments, the method further comprises inactivating the first and second target complexes prior to contacting the sample with a probe to label the third target and second detection reagents.

BRIEF DESCRIPTION OF THE FIGURES The patent or application file contains at least one drawing executed in

color. Copies of this patent or patent application publication with color drawings

will be provided to the Office upon request and the payment of the necessary fee.

FIG. 1A illustrates an embodiment of an unmasked hapten.

FIG. 1B illustrates an embodiment of a caged hapten.

FIG. 1C illustrates a structure of a caging group coupled to a hapten.

FIG. 2 is a schematic illustrating the interaction between an unmasking

enzyme-antibody conjugate comprising an alkaline phosphatase (bound to Target

2) and a caged hapten-antibody conjugate (bound to Target 1), where the

unmasking enzyme of the unmasking enzyme-antibody conjugate reacts with an

enzyme substrate portion of the caged hapten-antibody conjugate (by virtue of the

proximity of Target 1 and Target 2 to each other) to provide the respective

unmasked hapten, which may be detected.

FIG. 3 is a schematic illustrating an unmasking enzyme-antibody conjugate (bound to Target 2) and a caged hapten-antibody conjugate (bound to

Target 1) where the two targets are not in close proximity to each other, such that

the unmasking enzyme of the unmasking enzyme-antibody conjugate does not

interact with an enzyme substrate portion of the caged hapten-antibody conjugated,

and thus the caged hapten remains masked and unable to be detected.

FIG. 4 provides a flowchart illustrating the steps of detecting protein

dimers and/or total protein in a sample.

FIG. 5 is a schematic illustrating an embodiment of an IHC staining

protocol where a single antigen is detected with a secondary antibody labeled with

cHQ.

FIG. 6A is an image of the detection of PSA on prostate tissue with a

secondary antibody labeled with cHQ and treatment with AP.

FIG. 6B is an additional image of the lack of detection of PSA on prostate

tissue with a secondary antibody labeled with cHQ and no AP treatment (negative

control).

FIG. 6C is an additional image of the detection of PSA on prostate tissue

with a secondary antibody labeled with native HQ.

FIG. 7A is an image of total protein for Beta-Catenin as measured with

DAB staining.

FIG 7B is an image of proteins with positive proximity as measured with

DAB staining.

FIG 7C is an image of total protein for E-cadherin as measured with DAB

staining.

FIG. 8A is an image of total protein for EGFR as measured with DAB

staining on FFPE tonsil tissue.

FIG. 8B is an image of co-localized proteins without proximity as

measured with the absence of DAB staining on FFPE tonsil tissue.

FIG. 8C is an image of total protein for E-cadherin as measured with DAB

staining on FFPE tonsil tissue.

FIG. 9A is an image of co-localized proteins with proximity as measured

with DAB staining on FFPE breast tissue.

FIG. 9B is an additional image of co-localized proteins without proximity

as measured by the absence of DAB staining on FFPE breast tissue.

FIG. 10A is an image of signal intensity based on the number of caged

hapten labels on the secondary antibody.

FIG. 10B is an additional image of the increase in signal intensity based on

the increased number of caged hapten labels on the secondary antibody.

FIG. 11A is an image of proximity signal detection as measured with HRP

Silver staining.

FIG. 11B is an additional image of proximity signal detection as measured

with HRP-Purple chromogen staining.

FIG. 12 is a schematic illustrating multiplex detection of both proteins

(Target 1 and Target 2) in close proximity and total protein (Target 2).

FIG. 13A is an image demonstrating detection of proximal proteins with

HRP-DAB.

FIG. 13B is an image demonstrating detection of proximal proteins with

HRP-Silver and total protein with AP-Yellow.

FIG. 14 is an image demonstrating detection of the proximity signal using

HRP-Purple and total protein using AP-Yellow.

FIG. 15 illustrates the structures of caged haptens and specifies particular

enzymes which may react with the enzyme substrate portion of the caged hapten.

FIG. 16 provides an image of proximity signal detection of PD-1 and PD

Li proteins as measured with HRP-Purple chromogen staining in a FFPE non

small cell lung cancer (NSCLC) case (Arrows = PD-Li/PD-1 proximity signal).

FIG. 17 provides an image of proximity signal detection of PD-1 and PD

Li proteins as measured with HRP-Purple chromogen staining and total protein for

PD-1 with AP-Yellow in FFPE tonsil (Arrows = PD-Li/PD-1 proximity signal; Dashed circles = PD-1 protein signal).

FIG. 18 provides an image of a negative control for proximity signal

detection of PD-1 and PD-L1 proteins where the PD-1 antibody was omitted in

FFPE tonsil and no signal was observed.

FIG. 19 provides an image of multiplex staining with proximity signal

detection of PD-1 and PD-L1 proteins as measured with HRP-Purple chromogen

staining and CD8 with AP-Yellow in FFPE tonsil (Arrows = PD-Li/PD-1 proximity signal; Dashed circles = CD8 protein signal).

FIG. 20A is an image of total protein for Her2 as measured with DAB

staining.

FIG. 20B is an image of Her2/Her3 proteins with positive proximity as

measured with DAB staining.

FIG. 20C is an image of total protein for Her3 as measured with DAB

staining.

DETAILED DESCRIPTION

Disclosed herein are caged haptens and their method of synthesis. Also

disclosed herein are conjugates comprising a caged hapten. As will be described in

more detail herein, the caged hapten conjugates may be used to detect proximal

antigens in tissue samples. These and other embodiments are described herein.

Definitions

As used herein, the singular terms "a," "an," and "the" include plural

referents unless context clearly indicates otherwise. Similarly, the word "or" is

intended to include "and" unless the context clearly indicates otherwise. The term

"includes" is defined inclusively, such that "includes A or B" means including A,

B, or A and B.

The terms "comprising," "including," "having," and the like are used

interchangeably and have the same meaning. Similarly, "comprises," "includes,"

"has," and the like are used interchangeably and have the same meaning.

Specifically, each of the terms is defined consistent with the common United States

patent law definition of "comprising" and is therefore interpreted to be an open

term meaning "at least the following," and is also interpreted not to exclude

additional features, limitations, aspects, etc. Thus, for example, "a device having components a, b, and c" means that the device includes at least components a, b and c. Similarly, the phrase: "a method involving steps a, b, and c" means that the method includes at least steps a, b, and c. Moreover, while the steps and processes may be outlined herein in a particular order, the skilled artisan will recognize that the ordering steps and processes may vary.

As used herein, alkaline phosphatase (AP) is an enzyme that removes (by

hydrolysis) and transfers phosphate group organic esters by breaking the

phosphate-oxygen bond, and temporarily forming an intermediate enzyme

substrate bond. For example, AP hydrolyzes naphthol phosphate esters (a

substrate) to phenolic compounds and phosphates. The phenols couple to colorless

diazonium salts (chromogen) to produce insoluble, colored azo dyes.

As used herein, the term "antibody," occasionally abbreviated "Ab," refers

to immunoglobulins or immunoglobulin-like molecules, including by way of

example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations

thereof, and similar molecules produced during an immune response in any

vertebrate, (e.g., in mammals such as humans, goats, rabbits and mice) and

antibody fragments that specifically bind to a molecule of interest (or a group of

highly similar molecules of interest) to the substantial exclusion of binding to other

molecules. Antibody further refers to a polypeptide ligand comprising at least a

light chain or heavy chain immunoglobulin variable region which specifically

recognizes and binds an epitope of an antigen. Antibodies may be composed of a

heavy and a light chain, each of which has a variable region, termed the variable

heavy (VH) region and the variable light (VL) region. Together, the VH region and

the VL region are responsible for binding the antigen recognized by the antibody.

The term antibody also includes intact immunoglobulins and the variants and

portions of them well known in the art.

As used herein, the phrase "antibody conjugates," refers to those antibodies

conjugated (either directly or indirectly) to one or more labels, where the antibody

conjugate is specific to a particular target and where the label is capable of being

detected (directly or indirectly), such as with a secondary antibody (an anti-label

antibody). For example, an antibody conjugate may be coupled to a hapten such as

through a polymeric linker and/or spacer, and the antibody conjugate, by means of

the hapten, may be indirectly detected. As an alternative example, an antibody conjugate may be coupled to a chromogen, such as through a polymeric linker and/or spacer, and the antibody conjugate may be detected directly. Antibody conjugates are described further in US Publication No. 2014/0147906 and US

Patent Nos. 8,658,389; 8,686,122; 8,618,265; 8,846,320; and 8,445,191. By way of a further example, the term "antibody conjugates" includes those antibodies

conjugated to an enzyme, e.g. HRP or AP.

As used herein, the term "antigen" refers to a compound, composition, or

substance that may be specifically bound by the products of specific humoral or

cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can

be any type of molecule including, for example, haptens, simple intermediary

metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as

macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins.

As used herein, the term a "biological sample" can be any solid or fluid

sample obtained from, excreted by or secreted by any living organism, including

without limitation, single celled organisms, such as bacteria, yeast, protozoans, and

amoebas among others, multicellular organisms (such as plants or animals,

including samples from a healthy or apparently healthy human subject or a human

patient affected by a condition or disease to be diagnosed or investigated, such as

cancer). For example, a biological sample can be a biological fluid obtained from,

for example, blood, plasma, serum, urine, bile, ascites, saliva, cerebrospinal fluid,

aqueous or vitreous humor, or any bodily secretion, a transudate, an exudate (for

example, fluid obtained from an abscess or any other site of infection or

inflammation), or fluid obtained from a joint (for example, a normal joint or a joint

affected by disease). A biological sample can also be a sample obtained from any

organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or

can include a cell (whether a primary cell or cultured cell) or medium conditioned

by any cell, tissue or organ. In some examples, a biological sample is a nuclear

extract. In certain examples, a sample is a quality control sample, such as one of

the disclosed cell pellet section samples. In other examples, a sample is a test

sample. Samples can be prepared using any method known in the art by of one of

ordinary skill. The samples can be obtained from a subject for routine screening or

from a subject that is suspected of having a disorder, such as a genetic abnormality, infection, or a neoplasia. The described embodiments of the disclosed method can also be applied to samples that do not have genetic abnormalities, diseases, disorders, etc., referred to as "normal" samples. Samples can include multiple targets that can be specifically bound by one or more detection probes.

As used herein, the term "chromophore" refers to a molecule or a part of a

molecule (e.g. a chromogenic substrate) responsible for its color. Color arises

when a molecule absorbs certain wavelengths of visible light and transmits or

reflects others. A molecule having an energy difference between two different

molecular orbitals falling within the range of the visible spectrum may absorb

visible light and thus be aptly characterized as a chromophore. Visible light

incident on a chromophore may be absorbed thus exciting an electron from a

ground state molecular orbital into an excited state molecular orbital.

As used herein, the term "conjugate" refers to two or more molecules or

moieties (including macromolecules or supra-molecular molecules) that are

covalently linked into a larger construct. In some embodiments, a conjugate

includes one or more biomolecules (such as peptides, proteins, enzymes, sugars,

polysaccharides, lipids, glycoproteins, and lipoproteins) covalently linked to one or

more other molecules moieties.

As used herein, the terms "couple" or "coupling" refers to the joining,

bonding (e.g. covalent bonding), or linking of one molecule or atom to another

molecule or atom.

As used herein, the term "detectable moiety" refers to a molecule or

material that can produce a detectable (such as visually, electronically or

otherwise) signal that indicates the presence (i.e. qualitative analysis) and/or

concentration (i.e. quantitative analysis) of the label in a sample. A detectable

signal can be generated by any known or yet to be discovered mechanism

including absorption, emission and/or scattering of a photon (including radio

frequency, microwave frequency, infrared frequency, visible frequency and ultra

violet frequency photons). Examples of detectable moieties include chromogenic,

fluorescent, phosphorescent and luminescent molecules and materials.

As used herein, the term "epitopes" refers to an antigenic determinant, such

as continuous or non-continuous peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody binds to a particular antigenic epitope.

As used herein, horseradish peroxidase (HRP) is an enzyme that can be

conjugated to a labeled molecule. It produces a colored, fluorometric, or

luminescent derivative of the labeled molecule when incubated with a proper

substrate, allowing it to be detected and quantified. HRP acts in the presence of an

electron donor to first form an enzyme substrate complex and then subsequently

acts to oxidize an electronic donor. For example, HRP may act on 3,3'

diaminobenzidine hydrochloride (DAB) to produce a detectable color. HRP may

also act upon a labeled tyramide conjugate, or tyramide like reactive conjugates

(i.e. ferulate, coumaric, caffeic, cinnamate, dopamine, etc.), to deposit a colored or

fluorescent or colorless detectable moiety for tyramide signal amplification (TSA).

As used herein, the terms "multiplex," "multiplexed," or "multiplexing"

refer to detecting multiple targets in a sample concurrently, substantially

simultaneously, or sequentially. Multiplexing can include identifying and/or

quantifying multiple distinct nucleic acids (e.g., DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) both individually and in any and all combinations.

As used herein, the term "primary antibody" refers to an antibody which

binds specifically to the target protein antigen in a tissue sample. A primary

antibody is generally the first antibody used in an immunohistochemical procedure.

As used herein, the term "secondary antibody" herein refers to an antibody

which binds specifically to a primary antibody, thereby forming a bridge between

the primary antibody and a subsequent reagent (e.g. a label, an enzyme, etc.), if

any. The secondary antibody is generally the second antibody used in an

immunohistochemical procedure.

As used herein, the term "specific binding entity" refers to a member of a

specific-binding pair. Specific binding pairs are pairs of molecules that are

characterized in that they bind each other to the substantial exclusion of binding to

other molecules (for example, specific binding pairs can have a binding constant

that is at least 103 M-1 greater, 104 M-1 greater or 10 5 M-1 greater than a binding

constant for either of the two members of the binding pair with other molecules in

a biological sample). Particular examples of specific binding moieties include

specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A). Specific binding moieties can also include the molecules (or portions thereof) that are specifically bound by such specific binding proteins.

As used herein, the term "target" refers to any molecule for which the

presence, location and/or concentration is or can be determined. Examples of

target molecules include proteins, epitopes, nucleic acid sequences, and haptens,

such as haptens covalently bonded to proteins. Target molecules are typically

detected using one or more conjugates of a specific binding molecule and a

detectable label.

As used herein, the terms "tyramide signal amplification" or "TSA" refer to

an enzyme-mediated detection method that utilizes the catalytic activity of a

peroxidase (such as horseradish peroxidase) to generate high-density labeling of a

target molecule (such as a protein or nucleic acid sequence) in situ. TSA typically

involves three basic steps: (1) binding of a specific binding member (e.g., an

antibody) to the target followed by secondary detection of the specific binding

member with a second peroxidase-labeled specific binding member; (2) activation

of multiple copies of a labeled tyramide derivative (e.g., a hapten-labeled

tyramide) by the peroxidase; and (3) covalent coupling of the resulting highly

reactive tyramide radicals to residues (e.g., the phenol moiety of protein tyrosine

residues) proximal to the peroxidase-target interaction site, resulting in deposition

of haptens proximally (diffusion and reactivity mediated) to the target. In some

examples of TSA, more or fewer steps are involved; for example, the TSA method

can be repeated sequentially to increase signal. Methods of performing TSA and

commercial kits and reagents for performing TSA are available (see, e.g.,

AmpMap Detection Kit with TSA TM, Cat. No. 760-121, Ventana Medical Systems,

Tucson, Ariz.; Invitrogen; TSA kit No. T-20911, Invitrogen Corp, Carlsbad,

Calif.). Other enzyme-catalyzed, hapten or signaling linked reactive species can be

alternatively used as they may become available.

Caged Haptens In one aspect of the present disclosure are "caged haptens," such as

illustrated in FIG. 1B. As those of skill in the art will appreciate, haptens are small

molecules that anti-hapten antibodies have been raised against. A "caged hapten"

is a hapten whose structure has been modified such that a suitable anti-hapten antibody no longer recognizes the molecule and no binding event occurs. In effect, the hapten's identity is "masked" or "protected." The caged haptens disclosed herein have been designed with an enzyme cleavable cage such that the respective hapten, i.e. the un-caged or unmasked hapten, is released by enzymatic treatment to regenerate the native hapten (see, for example, FIG. 2, which illustrates the unmasking of a caged hapten via enzymatic treatment). Thus, in the presence of an appropriate enzyme, the caged hapten is unmasked and an anti-hapten antibody is free to bind to it.

In some embodiments, the caged haptens of the present disclosure have the

structure of Formula (I):

Y X Hapten- [A-]-Caging Group q (I), wherein

X is a bond, or a group comprising a branched or unbranched, substituted

carbon atoms, and optionally having one or more heteroatoms selected from the

group consisting of 0, N, or S;

A is a group comprising a branched or unbranched, substituted or

unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15

carbon atoms, and optionally having one or more heteroatoms selected from the

group consisting of 0, N, or S;

Y is a reactive group capable of forming a covalent bond with another

group; and

"hapten" and "Caging Group" are as described herein; and

q is 0 or 1.

In some embodiments, the caging group is an enzyme cleavable moiety

comprised of two covalently bonded components, namely (i) a leaving group

portion, and (ii) an enzyme substrate portion, as illustrated in Formula (IA).

Y X Hapten-f-A- Leaving Group-Enzyme Substrate q (IA).

In other embodiments, the caging group comprises only an enzyme

substrate, coupled directly or indirectly to the hapten as in Formula (IB):

Y X Hapten-f-A Enzyme Substrate q .. (IB).

Examples of suitable enzyme substrates (and the enzymes that act upon them)

include, but are not limited to, phosphate groups (acted upon by an alkaline

phosphatase), ester groups (acted upon by a lipase); sulfate groups (acted upon by

a sulfatase); glycoside groups (acted upon by a glycosylase); amide groups (acted

upon by an amidase or a protease); urea groups (acted upon by a urease); and nitro

groups (acted upon by a nitroreductase).

In some embodiments, the leaving group comprises a 5-, 6-, or 7-membered

aromatic ring or heterocyclic ring, where any position of the ring may be

substituted or unsubstituted. In some embodiments, the leaving group is a

substituted or unsubstituted 5-, 6-, or 7-membered heterocyclic ring having one,

two, or three heteroatoms selected from 0, N, or S. In some embodiments, the

leaving group is selected from a substituted or unsubstituted furan, pyrrole,

thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, pyridine, pyrazine,

pyrimidine, pyridazine, a triazine, 2H-pyran, 4H-pyran, 2H-thiopyran, 4H thiopyran, an oxazine, or a thiazine. In some embodiments, the 5-, 6-, or 7

membered aromatic or heterocyclic ring is substituted with a halogen, a -S-alkyl

group having between 1 and 4 carbon atoms; an -0-alkyl group having between 1

and 4 carbon atoms; a -N(H)-alkyl group having between 1 and 4 carbon atoms; a

-N-(alkyl) 2 group having between 1 and 6 carbon atoms; or a branched or

unbranched, substituted or unsubstituted alkyl group having between 1 and 4

carbon atoms which itself may comprise one or more heteroatoms selected from 0,

N, or S and/or which may be substituted with one or more halogens. In some embodiments, the leaving group may be substituted with a nitro group, a cyano group, or a carboxy group.

In some embodiments, the caging group has the structure of Formula (II):

R2

R1 R1

wherein

each R 1 is independently selected from H, F, Cl, Br, I, -0-methyl, -0-

ethyl, -O-n-propyl, -0--iso-propyl; -O-n-butyl, -0-sec-butyl, or -0-iso-butyl; a -S-alkyl group having between 1 and 4 carbon atoms; an -0-alkyl group having

between 1 and 4 carbon atoms; a -N(H)-alkyl group having between 1 and 4

group having between 1 and 4 carbon atoms and optionally substituted with N or

S; cyano groups; and carboxyl groups.

R2 is an enzyme substrate.

In some embodiments, each R g roup is the same. In other embodiments,

each R g roup is different (i.e. each R comprises a different moiety). For

example, a first Rg roup may comprise a halogen while a second R1 may comprise

an alkyl group.

While Formula (II) depicts a six-membered aromatic ring comprising six

carbon atoms, the skilled artisan will appreciate that one or more heteroatoms (e.g.

0, N, or S) may be substituted for one or more of the carbon atoms of the aromatic

ring.

In some embodiments, the ability of any leaving group to leave depends on

the electronics of the hapten. In some embodiments, the electronics of certain

haptens require electron donating groups to encourage leaving group ability. In

other embodiments, the haptens require an electron withdrawing group. For

example, depending on the pKa of the functional group being caged, some haptens

require the addition of electron donating groups on the leaving group of the caging group, while others may require electron withdrawing groups. Without wishing to be bound by any particular theory, is believed that hapten functional groups with a high pKa value tend to require electron donating groups. This is most likely because once the leaving group is expelled, the electron donating groups in this position are able to directly stabilize the resulting positive charge of the leaving group through inductive or resonance effects. An example of this was seen in the caged-HQ, where in the absence of electron donating groups, the leaving group would not leave without excessively high reaction temperature (about 100 °C). On the other hand, it is believed that for hapten functional groups with a low pKa value, electron withdrawing groups may be required. This is most likely because the once the leaving group is expelled, the resulting negative charge on the hapten functional group has such high stability that a destabilizing electronic force may be necessary on the leaving group to help stabilize the caged hapten against hydrolysis. It is believed that an electron withdrawing group could destabilize the positive charge on the leaving group, thereby making the caged hapten more stable toward hydrolysis.

The substituents R and R2 may be located at any position along the ring of

Formula (II) relative to the group coupling the Caging Group to the hapten. In

some embodiments, R 2 is positioned para to the group coupling the Caging Group

to the hapten, such as illustrated in Formula (IIA):

R2

(IIA). In other embodiments of the compounds of Formula (IIA), the substituents 1 2 R may independently be positioned ortho or meta to group R. In some g embodiments, each R roup of Formula (IIA) is different, i.e. each Rg roup

comprises a different moiety.

In further embodiments, the Caging Group has the structure of Formula

(JIB):

R2

(JIB).

In some embodiments, each R g roup of Formula (JIB) is different, i.e. each

R group comprises a different moiety.

In yet further embodiments, the Caging Group has the structure of Formula

(IIC):

Ri

R2 Ri

(IC). In some embodiments, each R g roup of Formula (IIC) is different, i.e.

each R g roup comprises a different moiety.

In yet further embodiments, the Caging Group has the structure of Formula

(IID):

R2

R1 R1

(IID). In some embodiments, each R group of Formula (IID) is different, i.e.

each R g roup comprises a different moiety.

Without wishing to be bound by any particular theory, it is believed that the

hydrolytic stability and leaving group ability of the caging group can be different

when coupled to different haptens. For example, the caging group with -OMe

groups installed at the meta position relative to the phosphate are believed to

provide a good mix of stability and reactivity on a 2-hydroxyquinoxaline (HQ)

hapten, but were believed to be comparatively less stable than desired when

installed on the nitropyrazole (NP) hapten. This was remedied by using a caging

group with -OMe groups installed ortho relative to the phosphate group. Again,

without wishing to be bound by any particular theory, it was believed that the 0

benzyl bond formed between the HQ hapten and the caging group was much more

stable than the N-benzyl bond formed between the NP and the caging group. This

was similarly remedied by introducing a -OMe group ortho to the benzyl group on

the HQ hapten to help "push" the electrons through the ring and break the bond,

once the phosphate was cleaved. The NP hapten was comparatively less stable, and

therefore it did not require the same "push."

Again, and without wishing to be bound by any particular theory, it is

believed that a caging group with no -OMe groups may require very high

temperatures to disassemble. However, a caging group with -OMe groups ortho to

the phosphate may require moderate temperature to disassemble; while a caging

group with the -OMe meta to the phosphate group may be able to disassemble at

room temperature. Without wishing to be bound by any particular theory, it is

believed that it may be possible to add stability while also increasing reactivity by adding one or two additional aliphatic groups to the benzyl position. It is believed that this may sterically protect the benzyl group from hydrolysis, while also creating a more stable 20 or 30 benzyl carbocation upon disassembly.

As noted herein, upon interaction of an enzyme with the enzyme substrate

portion of the caging group, the caging group undergoes an electronic change such

that the Caging Group separates from the caged hapten, resulting in an un-caged

hapten or un-masked hapten. FIG. 2 illustrates the electronic changes that occur

within a caged HQ hapten upon interaction of an alkaline phosphatase enzyme

with the phosphate enzyme substrate portion of the caging group. Similar

electronic changes occur in other caged hapten systems as will be appreciated by

those of ordinary skill in the art.

In some embodiments, haptens include, but are not limited to, pyrazoles

(e.g. nitropyrazoles); nitrophenyl compounds; benzofurazans; triterpenes; ureas

(e.g. phenyl ureas); thioureas (e.g. phenyl thioureas); rotenone and rotenone

derivatives; oxazole (e.g. oxazole sulfonamides); thiazoles (e.g. thiazole

sulfonamides); coumarin and coumarin derivatives; and cyclolignans. Additional

non-limiting examples of haptens include thiazoles; nitroaryls; benzofurans;

triperpenes; and cyclolignans.

Specific examples of haptens include di-nitrophenyl, biotin, digoxigenin,

and fluorescein, and any derivatives or analogs thereof. Other haptens are

described in United States Patent Nos. 8,846,320; 8,618,265; 7,695,929; 8,481,270; and 9,017,954, the disclosures of which are incorporated herein by

reference in their entirety.

As noted above, X may a bond; or a group comprising a branched or

unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group

having between 1 and 30 carbon atoms, and optionally having one or more

heteroatoms selected from the group consisting of 0, N, or S. In some

embodiments, X may comprise carbonyl, amine, ester, ether, amide, imine, thione

or thiol groups. In other embodiments, X may comprise one or more terminal

groups selected from an amine, a carbonyl, ester, ether, amide, imine, thione, or

thiol. In some embodiments, X has the structure of Formula (IIIA):

Ra

dQ -c- e (IIIA), wherein d and e are integers each independently ranging from 4 to 18; Q is a bond, 0, S, or N(R')(Rd); Ra and R are independently H, a C1 -C 4 alkyl group, F, Cl, or N(R )(Rd); and R' and Rd are independently CH 3 or H. In some

embodiments, d and e are integers each independently ranging from 1 to 24.

In other embodiments, the X has the structure depicted in Formula (IIB):

-CH - -Q

e (IIIB), wherein d and e are integers each independently ranging from 1 to 24; Q is a bond, 0, S, or N(R')(Rd); and R' and Rd are independently CH 3 or H. In other

embodiments, Q is 0. In yet other embodiments, the 'Linker'has the structure depicted in Formula

(IIIC):

CHr-

e e (IIIC), wherein d and e are integers each independently ranging from 1 to 24. In

some embodiments, d is 2 and e ranges from 2 through 24.

In yet other embodiments, X may include one or more alkylene oxide or

PEG groups (e.g. PEG2 through PEG24). A person of ordinary skill in the art will appreciate that, as the number of oxygen atoms increases, the hydrophilicity of the

compound also may increase.

Without wishing to be bound by any particular theory, it is believed that the

linker length may affect the proximity signal. For example, caged haptens

comprising PEG8, PEG4 and no PEG linkers were made and tested. The longer in

length the linker was, the more signal we saw on our control dimer system of E

cad and B-cat. While "more signal" may seem like it would always be useful, in

this case it was not because we also observed signal when testing on protein

markers that co-localize but are known to not form dimers (Ki67 and Bc6). We

found that using no PEG linker eliminated the signal on the co-localized control

system while still providing some signal on the known dimer control system. As a

result, some caged hapten embodiments do not include a PEG group.

As noted herein, A is a group comprising a branched or unbranched,

substituted or unsubstituted, saturated or unsaturated aliphatic group having

between 1 and 15 carbon atoms, and optionally having one or more heteroatoms

selected from the group consisting of 0, N, or S. In some embodiments, A has the

structure of Formula (IIID):

.Ra

c dRb

wherein dand eare integers each independently ranging from 2to 15; Qis a bond, 0, S, or N(R')(Rd); Ra and R are independently H, a C1 -C 4 alkyl group, F, Cl, or N(R )(Rd); and R' and Rd are independently CH 3 or H. In some

embodiments, d and e are integers each independently ranging from 1 to 12.

In some embodiments, the reactive group Y is a carbonyl-reactive group.

Suitable carbonyl-reactive groups include hydrazine, hydrazine derivatives, and

amine. In other embodiments, the reactive group Y is an amine-reactive group.

Suitable amine-reactive groups include active esters, such as NHS or sulfo-NHS,

isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals,

epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydrides and the like.

In yet embodiments, the reactive group Y is a thiol-reactive group. Suitable thiol

reactive groups include non-polymerizable Michael acceptors, haloacetyl groups

(such as iodoacetyl), alkyl halides, maleimides, aziridines, acryloyl groups, vinyl

sulfones, benzoquinones, aromatic groups that can undergo nucleophilic

substitution such as fluorobenzene groups (such as tetra and pentafluorobenzene

groups), and disulfide groups such as pyridyl disulfide groups and thiols activated

with Ellman's reagent.

Specific examples of caged haptens are provided below, including caged 7

(Diethylamino)coumarin-3-carboxylic acid (DCC), caged biotin, caged

nitropyrazole, caged thiazolesulfonamide (TS), and caged benzofurazan (BF).

Each of the caged haptens below comprise a substrate for an alkaline phosphatase

enzyme.

OH 0 HOf ,-0O 0Et N~ H 0 0 Et

O DCC 0

HOO H: H N- N

OH O H S H Biotin

a0

HO-P-OH 0 0 -_~O HO, , /

O OH

O 0 00 O~ O 0 CO C0202

NOO 02 O0 N H 0N NN H H 0 N- ;2N -- N '

0 0 OH HQ NP Fluorescein or FITC

HO-P -OH HO 0

OO

0 N. H '0 0 N~S-NN H o N\ TS BF

011 HO-P -OH 0

0,OH 0 .P\ N 0' OH 0 N 0 0 N ;N

H H HO

N 0

Ok N.O NX

-~ HO 0

NN NN N- N 0 H

011 HO-P-OH 0

N

N. N H 0 N; 0 H

HO '40 0

0

N NH 0 H Other examples of caged haptens, including specific enzymes that act upon

the enzyme substrate portion of the caged hapten, are provided in FIG. 15.

Synthesis of Caged Haptens The caged happens may be synthesized according to any methods known to

those of ordinary skill in the art. For example, a caging group may be prepared

starting with a substituted 4-hydroxybenzaldehyde. The hydroxyl group of the

substituted 4-hydroxybenzaldehyde may be substituted by a phosphate group,

followed by reduction of the aldehyde and substitution of the resulting benzyl

alcohol with a halogen through an Appel reaction. A caging group may then be

installed on the hapten of interest by reaction of the halogen-substituted caging

group with the hapten under basic conditions (if the hapten contains multiple

nucleophilic groups, it may require protection chemistry to select the desired

position for caging). The protected phosphate group of the caging group may then

be deprotected using trimethylsilyl bromide, followed by reaction of the caged

hapten with a reactive group (i.e maleimide) to facilitate conjugation with an

antibody.

The synthesis of a "Caged HQ Hapten" (see FIG. 1B) illustrated in

Example 1 and at Scheme 3. The synthesis of a "Caged NP Hapten" is illustrated

below in Scheme 1.

OH "_+_- 0 0 O. O CI O Os 1)NaBH4,THF O O

TEA, EtOAc 2) SOC2.

H 0 H 0 CI O p -O R

H H/ N-N 1)DSC,DMAP,DMF N- TMSBr 02N \l OH M- 02N- 0 2 2) H 2N -,.,NHBoc 2 NHBoc K2CO3, N -N CH2C2 TEA,DMF O0O 2N NHBoc 0

-O ,PO HOPl0 0 0 _0O HO 'O O OH <>-00 'K' NO 0 H 0 0A N-N TEA,DMF N-N 0 0 O2N N0 2N H

0 0 H

Schme1:Synheisof aCag,,-ed NPF Hatnsarted wit a subst"ituted 4~

Conjug~ates of Caged Haptens The present disclosure provides novel conjugates comprising a caged

hapten. In some embodiments, the caged hapten is conjugated to specific binding

entity, e.g. an antibody or nucleic acid probe. In other embodiments, the caged

hapten is conjugated to an antibody, as in Formula (IV):

Antibody Z- Hapten A-]-Caging Group] q n (IV), where

A is a group comprising a branched or unbranched, substituted or

unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15

carbon atoms, and optionally having one or more heteroatoms selected from the

group consisting of 0, N, or S;

Q is 0 or 1; n is an integer ranging from 1 to 25, and

Z is a bond, or a group comprising a branched or unbranched, substituted or

unsubstituted, saturated or unsaturated aliphatic group having between 1 and 30 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of 0, N, or S. In some embodiments, Z comprises a group having the structure of any of Formulas (IIIA), (IIIB), or (IIC). In some embodiments, n is an integer ranging from 1 to 8. In other embodiments, n is an integer ranging from 1 to 6. In yet other embodiments, n is an integer ranging from 1 to 4. In further embodiments, n is an integer ranging from 2 to 5. In yet further embodiments, n is 3. In even further embodiments, n is

4. In some embodiments, the caged haptens are conjugated to primary

antibodies (e.g. a caged hapten conjugated to an antibody specific for Beta

Catenin). In other embodiments, the caged haptens are conjugated to secondary

antibodies (e.g. a caged hapten conjugated to an antibody specific for an anti-Beta

Catenin antibody).

The caged haptens may be coupled to any portion of an antibody. Three

functional groups of antibodies suitable for covalent modifications include (i)

amines (-NH2), (ii) thiol groups (-SH), and (iii) carbohydrate residues. As such, any of the caged haptens disclosed herein may be coupled to amine residues, thiol

residues, and carbohydrate residues or any combination thereof. In some

embodiments, the caged haptens are coupled to Fc portions of the antibody.

Synthesis of Caged Hapten Conjugates The conjugates of the present disclosure may be synthesized according to

any means known to those of ordinary skill in the art.

In some embodiments, a caged hapten is conjugated to a thiol group of an

antibody. In some embodiments, thiol groups are first introduced to the antibody

by treating the antibody with a reducing agent such as dithiothreitol (DTT) or

dithioerythritol (DTE). For a mild reducing agent, such as DTE or DTT, a

concentration of between about 1 mM and about 40 mM (for example, a

concentration of between about 5 mM and about 30 mM or between about 15 mM

and about 25 mM) is utilized to introduce a limited number of thiols (such as

between about 2 and about 6) to the antibody, while keeping the antibody intact

(which can be determined by size-exclusion chromatography). Following

treatment with the reducing agent, an excess of a caged hapten bearing a thiol reactive group (e.g. a maleimide group) is introduced to form the respective caged hapten-antibody conjugate.

In other embodiments, a caged hapten is conjugated to a Fc portion of an

antibody. In some embodiments, an Fc portion of an antibody is first oxidized to

form an aldehyde and the caged hapten is subsequently coupled to the oxidized Fc

portion of the antibody through a reactive functional group on the caged hapten

(e.g. with a carbonyl-reactive group, such as hydrazide group).

In yet other embodiments, a caged hapten is conjugated to a lysine residue

of an antibody. As illustrated in the synthetic scheme which follows (Scheme 2),

in some embodiments, the antibody is first treated with an excess of Traut's reagent

(2-iminothiolane hydrochloride) before adding an excess of an appropriately

functionalized caged hapten (e.g. one bearing a thiol reactive group, such as a

maleimide group). 0 11 HO-P -OH 0

0 0

0 HO0 N~

N N N H 0 SHs 0 (D H H AbrNH2 ~ Ab' S N NH2 Ab N ma em HQe Caged Ab Cr 2 Ab Maleimirye Ab" Traut's Reagent

15 Schm 2 Synhetic metholo i r n th r n between aai group ~ ~ ~ ~~~~- In aanidy(A"wthTaut's Reae\" oloe'ycopigo

Following synthesis, the conjugates may be purified, such as by size

exclusion chromatography (SEC), and then characterized, such as by gel

electrophoresis and/or UV-Vis.

Detection of Caged Hapten Antibody Conjug~ates In some embodiments, detection reagents are utilized to enable detection of

caged hapten conjugates, or complex of a caged hapten conjugate and a target. In

some embodiments, the detection reagents employed are specific to the respective unmasked hapten corresponding to the caged hapten of any caged hapten conjugate. Thus, the terms "respective unmasked hapten" or "unmasked hapten" refer to caged haptens that have been "un-caged" with an appropriate unmasking enzyme to reveal the "native" hapten, i.e. an unmasking enzyme that is reactive with an enzyme substrate portion of the caged hapten. The steps of "un-caging" or

"unmasking" are described further herein and depicted at least in FIG. 2. As will

be described herein, detection reagents may also include components designed to

increase signal, e.g. signal amplification components or signal amplification kits.

In some embodiments, the detection reagents specific to the unmasked

hapten are secondary antibodies specific to the unmasked hapten of the caged

hapten conjugate, i.e. anti-unmasked hapten antibodies, and are themselves

conjugated to a detectable moiety. A "detectable moiety" is a molecule or material

that can produce a detectable (such as visually, electronically or otherwise) signal

that indicates the presence (i.e. qualitative analysis) and/or concentration (i.e.

quantitative analysis) of the caged hapten-antibody conjugate and/or unmasking

enzyme-antibody conjugate in a sample. A detectable signal can be generated by

any known or yet to be discovered mechanism including absorption, emission

and/or scattering of a photon (including radio frequency, microwave frequency,

infrared frequency, visible frequency and ultra-violet frequency photons).

In some embodiments, the detectable moiety of the anti-unmasked hapten

antibody includes chromogenic, fluorescent, phosphorescent and luminescent

molecules and materials, catalysts (such as enzymes) that convert one substance

into another substance to provide a detectable difference (such as by converting a

colorless substance into a colored substance or vice versa, or by producing a

precipitate or increasing sample turbidity), haptens that can be detected through

antibody-hapten binding interactions using additional detectably labeled antibody

conjugates, and paramagnetic and magnetic molecules or materials. Of course, the

detectable moieties can themselves also be detected indirectly, e.g. if the detectable

moiety is a hapten, then yet another antibody specific to that detectable moiety

may be utilized in the detection of the detectable moiety, as known to those of

ordinary skill in the art.

In some embodiments, the anti-unmasked hapten antibody includes a

detectable moiety selected from the group consisting of Cascade Blue acetyl azide;

Dapoxylsulfonic acid/carboxylic acid DY- 405; Alexa Fluor 405 Cascade Yellow

pyridyloxazole succinimidyl ester (PyMPO); Pacific Blue DY- 415; 7 hydroxycoumarin-3-carboxylic acid DYQ-425; 6-FAM phosphoramidite; Lucifer Yellow; Alexa Fluor 430 Dabcyl NBD chloride/fluoride; QSY 35 DY-485XL; Cy2 DY-490; Oregon Green 488 Alexa Fluor 488 BODIPY 493/503 C3 DY 480XL; BODIPY FL C3 BODIPY FL C5 BODIPY FL-X DYQ-505; Oregon Green 514 DY-510XL; DY-481XL; 6-carboxy- 4',5'-dichloro-2',7' dimethoxyfluorescein succinimidyl ester (JOE); DY-520XL; DY-521XL; BODIPY R6G C3 erythrosin isothiocyanate; 5- carboxy-2',4',5',7' tetrabromosulfonefluorescein Alexa Fluor 532 6-carboxy-2',4,4',5'7,7'

hexachlorofluorescein succinimidyl ester (HEX); BODIPY 530/550 C3 DY-530; BODIPY TMR-X DY-555; DYQ-1; DY-556; Cy3 DY-547; DY-549; DY-550; Alexa Fluor 555 Alexa Fluor 546 DY-548; BODIPY 558/568 C3 Rhodamine red-X QSY 7 BODIPY 564/570 C3 BODIPY 576/589 C3 carboxy-X-rhodamine (ROX); Alexa Fluor 568 DY-590; BODIPY 581/591 C3 DY-591; BODIPY TR X Alexa Fluor 594 DY-594; carboxynaphthofluorescein DY-605; DY-610; Alexa Fluor 610 DY-615; BODIPY 630/650-X erioglaucine; Alexa Fluor 633 Alexa Fluor 635 succinimidyl ester,; DY-634; DY-630; DY-631 ; DY-632; DY 633; DYQ-2; DY-636; BODIPY 650/665-X DY-635; Cy5 Alexa Fluor 647 DY 647; DY-648; DY- 650; DY-654; DY-652; DY-649; DY-651; DYQ-660; DYQ 661; Alexa Fluor 660 Cy5.5 DY-677; DY-675; DY-676; DY-678; Alexa Fluor 680 DY-679; DY-680; DY-682; DY-681 ; DYQ-3; DYQ-700; Alexa Fluor 700 DY-703; DY-701; DY-704; DY- 700; DY-730; DY-731; DY-732; DY-734; DY 750; Cy7 DY- 749; DYQ-4; and Cy7.5. Fluorophores belong to several common chemical classes including

coumarins, fluoresceins (or fluorescein derivatives and analogs), rhodamines,

resorufins, luminophores and cyanines. Additional examples of fluorescent

molecules can be found in Molecular Probes Handbook - A Guide to Fluorescent

Probes and Labeling Technologies, Molecular Probes, Eugene, OR, ThermoFisher

Scientific, 11 Edition. In other embodiments, the fluorophore is selected from

xanthene derivatives, cyanine derivatives, squaraine derivatives, naphthalene

derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives,

pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In other embodiments, the fluorescent moiety is selected from a CF dye (available from Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY (available from Invitrogen), Alexa

Fluor (available from Invitrogen), DyLight Fluor (e.g. DyLight 649) (available from Thermo Scientific, Pierce), Atto and Tracy (available from Sigma Aldrich),

FluoProbes (available from Interchim), Abberior Dyes (available from Abberior),

DY and MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (available

from Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau and Square

Dyes (available from SETA BioMedicals), Quasar and Cal Fluor dyes (available

from Biosearch Technologies), SureLight Dyes (available from APC, RPEPerCP,

Phycobilisomes) (Columbia Biosciences), and APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

In other embodiments, the anti-unmasked hapten antibody is conjugated to

an enzyme. In these embodiments, the final proximity signal can be generated

with any enzyme conjugated to the relevant anti-unmasked hapten antibody, with

the exception of the enzyme that is used for unmasking (e.g. an unmasking enzyme

of an unmasking enzyme-antibody conjugate, described further herein). In some

embodiments, suitable enzymes include, but are not limited to, horseradish

peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, neuramindase, -galactosidase, -glucuronidase or -lactamase. In other

embodiments, enzymes include oxidoreductases or peroxidases (e.g. HRP). In

these embodiments, the enzyme conjugated to the anti-unmasked hapten antibody

catalyzes conversion of a chromogenic substrate, a covalent hapten, a covalent

fluorophore, non-covalent chromogens, and non-covalent fluorophores to a

reactive moiety which labels a sample proximal to or directly on the target.

Particular non-limiting examples of chromogenic compounds/substrates

include diaminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), BCIP/NBT, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2'-azino-di-[3 ethylbenzothiazoline sulphonate] (ABTS), o -dianisidine, 4-chloronaphthol (4

CN), nitrophenyl- 0-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5 bromo-4-chloro-3-indolyl- -galactopyranoside (X-Gal), methylumbelliferyl--D galactopyranoside (MU-Gal), p-nitrophenyl- a-D-galactopyranoside (PNP), 5- bromo-4-chloro-3-indolyl- 0-D-glucuronide (X-Gluc), 3-amino-9-ethyl carbazole

(AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue, and tetrazolium

violet. DAB, which is oxidized in the presence of peroxidase and hydrogen

peroxide, results in the deposition of a brown, alcohol-insoluble precipitate at the

site of enzymatic activity.

In some embodiments, the chromogenic substrates are signaling conjugates

which comprise a latent reactive moiety and a chromogenic moiety. In some

embodiments, the latent reactive moiety of the signaling conjugate is configured to

undergo catalytic activation to form a reactive species that can covalently bond

with the sample or to other detection components. The catalytic activation is

driven by one or more enzymes (e.g., oxidoreductase enzymes and peroxidase

enzymes, like horseradish peroxidase) and results in the formation of a reactive

species. These reactive species are capable of reacting with the chromogenic

moiety proximal to their generation, i.e. near the enzyme. Specific examples of

signaling conjugates are disclosed in US Patent Publication No. 2013/0260379, the

disclosure of which is hereby incorporated by reference herein in its entirety.

Other substrates include those set forth in U.S. Patent No. 5,583,001, U.S.

application publication No. 2012/0171668, and PCT/EP2015/0533556, the disclosures of which are hereby incorporate by reference herein in their entireties.

Suitable chromogenic substrates or fluorescent substrates coupled to TSA or QM

conjugates, as noted in the above incorporated references, include N,N'

biscarboxypentyl-5,5'-disulfonato-indo-dicarbocyanine (Cy5), 4-(dimethylamino)

azobenzene-4'-sulfonamide (Dabsyl), tetramethylrhodamine (Tamra), and

Rhodamine 110 (Rhodamine). In some embodiments, the chromogenic substrates, fluorescent substrates,

or signaling conjugates are selected such that peak detectable wavelengths of any

chromogenic moiety do not overlap with each other and are readily detectable by a

pathologist or an optical detector (e.g. a scanner). In some embodiments, the

chromogenic moieties are selected such that the peak wavelengths of the different

chromogenic moieties are separated by at least about 50nm. In other

embodiments, the chromogenic moieties are selected such that the peak

wavelengths of the different chromogenic moieties are separated by at least about

70nm. In yet other embodiments, the chromogenic moieties are selected such that the peak wavelengths of the different chromogenic moieties are separated by at least about 100nm.

In yet further embodiments, the chromogenic moieties are selected such

that the chromogenic moieties, when introduced to the tissue specimen, provide for

different colors (e.g. yellow, blue, magenta). In some embodiments, the

chromogenic moieties are selected such that they provide a good contrast between

each other, e.g. a separation of colors that are optically recognizable. In some

embodiments, the chromogenic moieties are selected such that when placed in

close proximity of each other provide for a signal or color that is different than the

signals or colors of either of the chromogenic moieties when observed alone.

Proximity Detection Using! Caged Hapten Conjug~ates As will be described in more detail herein, the present disclosure enables

the detection of protein dimers or proteins in close proximity to each other. Non

limiting examples of protein-protein interactions include any of the Herl/2/3/4

proteins with each other; PD-1 with PD-Li; and/or PD-L2, EGFR (Herl) with any of it associated ligands (AREG, EREG). Without wishing to be bound by any particular theory, it is believed that the

disclosed proximity assay is more general than merely measuring protein-protein

interactions. Indeed, the disclosed assay allows for the measurement of the

proximity of binding moieties. In practice, the binding moieties (e.g. antibodies)

may be directed against targets with minimal or no distance between them.

Examples of this could include signaling events like phosphorylation of proteins.

In this case, if one antibody is directed against an epitope on a protein (e.g. HER2),

and a second antibody is directed against all phospho-tyrosines, then the proximity

signal would represent all the phosphorylated HER2 proteins. This type of assay is

more binary (yes/no) than pairs of proteins that interact with each other.

In some embodiments, the assay is able to detect protein dimers or proteins

having a proximity of 5000nm or less. In other embodiments, the assay is able to

detect protein dimers or proteins having a proximity of 2500nm or less. In yet

other embodiments, the assay is able to detect protein dimers or proteins having a

proximity of 1000nm or less. In further embodiments, the assay is able to detect

protein dimers or proteins having a proximity of 500nm or less.

The skilled artisan will appreciate that the caged hapten conjugates may be

used in both simplex assays (detection of protein dimers or protein proximity) and

multiplex assays (detection of protein dimers or protein proximity and detection of

total protein). "Total protein" refers to the normal IHC visualization of any given

protein, whereas a proximity signal is the portion of this protein that is involved in

a given interaction. For example, and in the case of a PD-1/PD-L1 assay, the

proximity signal would visualize only the interaction between PD-1 and PD-Li,

whereas the total protein signal would visualize all PD-1 in the sample. Expressing

the score for proximity as a numerator and the score for total protein as a

denominator could give the fraction or percentage of PD-1 that is involved in an

interaction. This may be important as a diagnostic for detecting active

pharmaceutical ingredients that disturb protein-protein interaction where the

expression of protein is less important that the number of interacting proteins. This

is believed to hold true for phosphorylation, as described above, where instead of

just receiving an arbitrary score for the phosphorylated signal, one may be able to

quantify what percentage of a give protein is phosphorylated.

With reference to FIG. 4, the detection of protein dimers takes place in two

general stages. In a first stage, a sample is labeled with an antibody conjugates at

step 150. In a second stage, the sample is contacted with first detection reagents,

and optionally second detection reagents, at step 160.

In some embodiments, a sample is first contacted at step 100 with an

unmasking enzyme-antibody conjugate specific for a target, to form a target

unmasking enzyme-antibody conjugate complex. Subsequently, the sample is then

contacted at step 110 with a caged hapten-antibody conjugate, such as those of

Formula (III), and specific for another target, to form a target-caged hapten

antibody conjugate complex. As will be appreciated by the skilled artisan, an

unmasking enzyme is selected that is reactive with an enzyme substrate portion of

the caged hapten-antibody conjugate. The skilled artisan will also appreciate that

steps 100 and 110 may be performed in any order or may be performed

simultaneously. In some embodiments, a reversible enzyme inhibitor is also

introduced prior to or simultaneously with the introduction of the caged hapten

antibody conjugate. For example, in some embodiments, the caged hapten

antibody conjugate may be formulated with a phosphate buffer.

As noted herein, the caged hapten portion of the caged hapten-antibody

conjugate is capable of becoming unmasked to provide the respective unmasked

hapten, i.e. the native hapten. As illustrated in FIG. 2, if a first target 101 is in

sufficient proximity to a second target 102, the caged hapten-antibody conjugate

103A will be provided in proximity (the proximity being labeled 105) to the unmasking enzyme-antibody conjugate 104 such that the unmasking enzyme of the

unmasking enzyme-antibody conjugate 104 may react with the enzyme substrate of

the caged hapten-antibody conjugate 103A. This in turn results in the formation of

a first target unmasked hapten-antibody conjugate complex (103B). As illustrated

in FIG. 2, the first target unmasked hapten-antibody conjugate complex (103B) is

able to bind or be recognized by other specific binding entities (e.g. a secondary

antibody 106). In some embodiments, the method also includes one or more "decaging

steps" where on-slide conditions are changed to enhance enzyme activity. During

the antibody conjugate binding portion of the assay, when the conjugates are in

excess, steps are taken to prevent incidental "decaging" of the caged hapten which

would lead to false positive results. These steps include adding reversible enzyme

inhibitors (e.g. to prevent the action of the enzyme on the caging group). For

example, in the context of alkaline phosphatase (AP), these inhibitors can include

phosphate, phenylalanine and EDTA which are believed to be able to reduce the

enzyme activity by different mechanisms. In some embodiments, non-bound

antibody conjugates are removed by washing, it is then necessary to change the on

slide conditions to be favorable for the optimal enzyme activity to allow

"decaging" to occur. Each decaging enzyme will have its own optimal conditions

(buffers, salts, cofactors, temperature). These "decaging steps" including any of

washing steps, steps to change the pH of the solutions or reagents present on the

slide, the addition of cofactors, or the changing of temperature (e.g. temperatures

ranging from about 37C to about 50C) are chosen to enhance the activity of the

enzyme and promote "decaging," without interfering with the specific binding of

the antibody conjugates. For example, and in the case of AP, washes are used to

remove residual inhibitors (e.g. phosphate), buffers are also used to increase the

activity (e.g. Tris, to adjust the pH to greater than about 8), and cofactors (e.g.

magnesium) are added. In some embodiments, each of these conditions are optimized in the context of the entire assay, for example the activity of AP is enhanced by the addition of magnesium ions ranging in concentration from 1 mM to 1 M. However at concentrations of magnesium ions greater than 100 mM the antibody binding is affected, so this limits the amount that may be added. Another example of an optimizable step is the temperature of the "decaging" steps. The

"decaging" event after the enzymatic step is driven by thermodynamics and can be

accelerated by heating. However, temperatures greater than 60 °C can negatively

affect both the enzyme activity and the antibody conjugate binding.

On the other hand, and as illustrated in FIG. 3, if a first target 101 is not in

103A will not be provided in proximity (the proximity being labeled 108) to the unmasking enzyme-antibody conjugate 104. In this instance, the unmasking

enzyme will not be reactive with the enzyme substrate of the caged hapten

antibody conjugate 103A, and thus the caged hapten will remain in a masked or

protected state, i.e. it is not capable of binding or being recognized by other

specific binding entities.

Referring to FIGs. 2, 4, and 12, in some embodiments, the sample is then

contacted at step 120 with first detection reagents (106), the first detection reagents

being specific to the unmasked hapten of the first target unmasked hapten-antibody

conjugate complex (103B). In some embodiments, the first detection reagents

include a secondary antibody (106) specific for the unmasked hapten (103B),

namely an anti-unmasked hapten antibody. In some embodiments, the anti

unmasked hapten antibody (106) is conjugated to a detectable moiety (e.g. in FIGs.

2 and 12, the detectable moiety is a HRP enzyme, where the HRP enzyme acts

upon a substrate, such as a silver chromogenic substrate (111)). The skilled artisan

will, of course, appreciate that the first detection reagents (106) will only bind if

the native or unmasked hapten (103B) of the first target unmasked hapten-antibody

conjugate complex is revealed by the unmasking enzyme of the unmasking

enzyme-antibody conjugate (104). Thus, signal (107) from the detectable moiety

of the first detection reagents (106) will only be able to be detected at step 140 if

the first and second targets (101 and 102), and, hence, the antibody conjugates

(103A and 104), are in close proximity to each other. Here, detected signal (107)

is representative of a protein dimer or proteins/targets in close proximity.

In some embodiments, an amplification step may be carried out to increase

detectable signal. For example, amplification components may be introduced to

further label the unmasked hapten of the first target unmasked hapten-antibody

conjugate with additional reporter moieties, e.g. additional haptens or other

"detectable moieties." By way of example, an anti-unmasked hapten antibody

conjugated to an amplification hapten (or, in other embodiments, conjugated to an

enzyme) may be introduced to label the unmasked hapten of the first target

unmasked hapten-antibody conjugate with a plurality of amplification haptens.

Subsequently, anti-amplification hapten antibodies, each conjugated to a detectable

moiety, may be introduced. In some embodiments, the anti-amplification hapten

antibodies are conjugated to an enzyme, where the enzyme acts upon an introduced

substrate to produce a signal (e.g. a chromogenic substrate or a fluorescent

substrate to produce a visual signal). TSA and QM conjugates, each described

herein, may be used in any amplification step. In some examples, signal

amplification is carried out using OPTIVIEW Amplification Kit (Ventana Medical

Systems, Inc., Tucson, Ariz., Catalog No. 760-099).

The skilled artisan will appreciate that the unmasking enzyme of the

unmasking enzyme-antibody conjugate may serve two functions, namely (i) to

unmask or reveal a caged hapten; and (ii) to react with another substrate (e.g. a

chromogenic substrate or a fluorescent substrate) such that a signal independent

from that generated by the unmasked hapten (i.e. the unmasked hapten-antibody

conjugate complex) may be detected. Accordingly, the presently disclosed system

allows for the proximity between two proteins to be visualized within the context

of the total protein stain for one of the proteins. Without wishing to be bound by

any particular theory, it is believed that the ability to multiplex proximity detection

within the context of another protein stain is a feature that allows for the possibility

of having a speedy, guided slide read (i.e. only looking for proximity signal within

the total protein) or the ability to quantitate the percentage of protein that is

interacting with another (a method of scoring the proximity assay).

Referring again to FIGs. 2, 4, and 12, following the introduction of the first

detection reagents (106), second detection reagents including a second detectable

moiety (109) may optionally be introduced to the sample at step 130 such that total

protein may be detected. In some embodiments, the second detectable moiety provides signals (112) different from that of the first detectable moiety (107). In some embodiments, the second detectable moiety comprises a substrate for the unmasking enzyme, e.g. a chromogenic substrate that provides yellow signals

(109). In other embodiments, the second detectable moiety comprises a signaling

conjugate. The skilled artisan will also appreciate that steps 120 and 130 may be

performed in any order or may be performed simultaneously.

In some embodiments, the biological samples are pre-treated with an

enzyme inactivation composition to substantially or completely inactivate

endogenous peroxidase activity. For example, some cells or tissues contain

endogenous peroxidase. Using an HRP conjugated antibody may result in high,

non-specific background staining. This non-specific background can be reduced by

pre-treatment of the sample with an enzyme inactivation composition as disclosed

herein. In some embodiments, the samples are pre-treated with hydrogen peroxide

only (about 1% to about 3% by weight of an appropriate pre-treatment solution) to

reduce endogenous peroxidase activity. Once the endogenous peroxidase activity

has been reduced or inactivated, detection kits may be added, followed by

inactivation of the enzymes present in the detection kits, as provided above. The

disclosed enzyme inactivation composition and methods can also be used as a

method to inactivate endogenous enzyme peroxidase activity.

In some embodiments if the specimen is a sample embedded in paraffin, the

sample can be deparaffinized using appropriate deparaffinizing fluid(s). After a

waste remover removes the deparaffinizing fluid(s), any number of substances can

be successively applied to the specimen. The substances can be for pretreatment

(e.g., protein-crosslinking, expose nucleic acids, etc.), denaturation, hybridization,

washing (e.g., stringency wash), detection (e.g., link a visual or marker molecule to

a probe), amplifying (e.g., amplifying proteins, genes, etc.), counterstaining,

coverslipping, or the like.

Automation The assays and methods of the present disclosure may be automated and

may be combined with a specimen processing apparatus. The specimen processing

apparatus can be an automated apparatus, such as the BENCHMARK XT

instrument and SYMPHONY instrument sold by Ventana Medical Systems, Inc.

Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S.

Pat. Nos. 5,650,327, 5,654,200, 6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. Published Patent Application Nos. 20030211630 and 20040052685, each of which is incorporated herein by reference in its entirety. Alternatively,

specimens can be manually processed.

The specimen processing apparatus can apply fixatives to the specimen.

Fixatives can include cross-linking agents (such as aldehydes, e.g., formaldehyde,

paraformaldehyde, and glutaraldehyde, as well as non-aldehyde cross-linking

agents), oxidizing agents (e.g., metallic ions and complexes, such as osmium

tetroxide and chromic acid), protein-denaturing agents (e.g., acetic acid, methanol,

and ethanol), fixatives of unknown mechanism (e.g., mercuric chloride, acetone,

and picric acid), combination reagents (e.g., Carnoy's fixative, methacarn, Bouin's

fluid, B5 fixative, Rossman's fluid, and Gendre's fluid), microwaves, and

miscellaneous fixatives (e.g., excluded volume fixation and vapor fixation).

If the specimen is a sample embedded in paraffin, the sample can be

deparaffinized with the specimen processing apparatus using appropriate

deparaffinizing fluid(s). After the waste remover removes the deparaffinizing

fluid(s), any number of substances can be successively applied to the specimen.

The substances can be for pretreatment (e.g., protein-crosslinking, expose nucleic

acids, etc.), denaturation, hybridization, washing (e.g., stringency wash), detection

(e.g., link a visual or marker molecule to a probe), amplifying (e.g., amplifying

proteins, genes, etc.), counterstaining, coverslipping, or the like.

The specimen processing apparatus can apply a wide range of substances to

the specimen. The substances include, without limitation, stains, probes, reagents,

rinses, and/or conditioners. The substances can be fluids (e.g., gases, liquids, or

gas/liquid mixtures), or the like. The fluids can be solvents (e.g., polar solvents,

non-polar solvents, etc.), solutions (e.g., aqueous solutions or other types of

solutions), or the like. Reagents can include, without limitation, stains, wetting

agents, antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.), antigen recovering fluids (e.g., aqueous- or non-aqueous-based antigen retrieval

solutions, antigen recovering buffers, etc.), or the like. Probes can be an isolated

nucleic acid or an isolated synthetic oligonucleotide, attached to a detectable label.

Labels can include radioactive isotopes, enzyme substrates, co-factors, ligands,

chemiluminescent or fluorescent agents, haptens, and enzymes.

After the specimens are processed, a user can transport specimen-bearing

slides to the imaging apparatus. The imaging apparatus used here is a brightfield

imager slide scanner. One brightfield imager is the iScan Coreo TM brightfield

scanner sold by Ventana Medical Systems, Inc. In automated embodiments, the

imaging apparatus is a digital pathology device as disclosed in International Patent

Application No.: PCT/US2010/002772 (Patent Publication No.: WO/2011/049608) entitled IMAGING SYSTEM AND TECHNIQUES or disclosed in U.S. Patent Application Publication No. 2014/0178169, filed on February 3, 2014, entitled IMAGING SYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. International Patent Application No. PCT/US2010/002772 and U.S. Patent

Application Publication No. 2014/0178169 are incorporated by reference in their

entities. In other embodiments, the imaging apparatus includes a digital camera

coupled to a microscope.

Counterstaining

Counterstaining is a method of post-treating the samples after they have

already been stained with agents to detect one or more targets, such that their

structures can be more readily visualized under a microscope. For example, a

counterstain is optionally used prior to coverslipping to render the

immunohistochemical stain more distinct. Counterstains differ in color from a

primary stain. Numerous counterstains are well known, such as hematoxylin, eosin,

methyl green, methylene blue, Giemsa, Alcian blue, and Nuclear Fast Red. DAPI

(4',6-diamidino-2-phenylindole) is a fluorescent stain that may be used.

In some examples, more than one stain can be mixed together to produce

the counterstain. This provides flexibility and the ability to choose stains. For

example, a first stain, can be selected for the mixture that has a particular attribute,

but yet does not have a different desired attribute. A second stain can be added to

the mixture that displays the missing desired attribute. For example, toluidine blue,

DAPI, and pontamine sky blue can be mixed together to form a counterstain.

Detection and/or Imaging!

Certain aspects, or all, of the disclosed embodiments can be automated, and

facilitated by computer analysis and/or image analysis system. In some

applications, precise color or fluorescence ratios are measured. In some

embodiments, light microscopy is utilized for image analysis. Certain disclosed

embodiments involve acquiring digital images. This can be done by coupling a

digital camera to a microscope. Digital images obtained of stained samples are

analyzed using image analysis software. Color or fluorescence can be measured in

several different ways. For example, color can be measured as red, blue, and green

values; hue, saturation, and intensity values; and/or by measuring a specific

wavelength or range of wavelengths using a spectral imaging camera. The samples

also can be evaluated qualitatively and semi-quantitatively. Qualitative assessment

includes assessing the staining intensity, identifying the positively-staining cells

and the intracellular compartments involved in staining, and evaluating the overall

sample or slide quality. Separate evaluations are performed on the test samples and

this analysis can include a comparison to known average values to determine if the

samples represent an abnormal state.

Kits In some embodiments, the caged hapten conjugates of the present

disclosure may be utilized as part of a "detection kit." In general, any detection kit

may include one or more caged hapten conjugates and detection reagents for

detecting the one or more caged hapten conjugates.

The detection kits may include a first composition comprising a caged

hapten conjugate and a second composition comprising detection reagents specific

to the first composition, such that the caged hapten conjugate may be detected via

the detection kit. In some embodiments, the detection kit includes a plurality of

caged hapten conjugates (such as mixed together in a buffer), where the detection

kit also includes detection reagents specific for each of the plurality of caged

hapten conjugates.

Of course, any kit may include other agents, including buffers;

counterstaining agents; enzyme inactivation compositions; deparafinization

solutions, etc. as needed for manual or automated target detection. The kit may

also include instructions for using any of the components of the kit, including methods of applying the kit components to a tissue sample to effect detection of one or more targets therein.

Samples and Targets Samples include biological components and generally are suspected of

including one or more target molecules of interest. Target molecules can be on the

surface of cells and the cells can be in a suspension, or in a tissue section. Target

molecules can also be intracellular and detected upon cell lysis or penetration of

the cell by a probe. One of ordinary skill in the art will appreciate that the method

of detecting target molecules in a sample will vary depending upon the type of

sample and probe being used. Methods of collecting and preparing samples are

known in the art.

Samples for use in the embodiments of the method and with the

composition disclosed herein, such as a tissue or other biological sample, can be

prepared using any method known in the art by of one of ordinary skill. The

samples can be obtained from a subject for routine screening or from a subject that

is suspected of having a disorder, such as a genetic abnormality, infection, or a

neoplasia. The described embodiments of the disclosed method can also be applied

to samples that do not have genetic abnormalities, diseases, disorders, etc., referred

to as "normal" samples. Such normal samples are useful, among other things, as

controls for comparison to other samples. The samples can be analyzed for many

different purposes. For example, the samples can be used in a scientific study or

for the diagnosis of a suspected malady, or as prognostic indicators for treatment

success, survival, etc.

Samples can include multiple targets that can be specifically bound by a

probe or reporter molecule. The targets can be nucleic acid sequences or proteins.

Throughout this disclosure when reference is made to a target protein it is

understood that the nucleic acid sequences associated with that protein can also be

used as a target. In some examples, the target is a protein or nucleic acid molecule

from a pathogen, such as a virus, bacteria, or intracellular parasite, such as from a

viral genome. For example, a target protein may be produced from a target nucleic

acid sequence associated with (e.g., correlated with, causally implicated in, etc.) a

disease.

A target nucleic acid sequence can vary substantially in size. Without

limitation, the nucleic acid sequence can have a variable number of nucleic acid

residues. For example, a target nucleic acid sequence can have at least about 10

nucleic acid residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues.

Similarly, a target polypeptide can vary substantially in size. Without limitation,

the target polypeptide will include at least one epitope that binds to a peptide

specific antibody, or fragment thereof. In some embodiments that polypeptide can

include at least two epitopes that bind to a peptide specific antibody, or fragment

thereof.

In specific, non-limiting examples, a target protein is produced by a target

nucleic acid sequence (e.g., genomic target nucleic acid sequence) associated with

a neoplasm (for example, a cancer). Numerous chromosome abnormalities

(including translocations and other rearrangements, amplification or deletion) have

been identified in neoplastic cells, especially in cancer cells, such as B cell and T

cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and

the like. Therefore, in some examples, at least a portion of the target molecule is

produced by a nucleic acid sequence (e.g., genomic target nucleic acid sequence)

amplified or deleted in at least a subset of cells in a sample.

Oncogenes are known to be responsible for several human malignancies.

For example, chromosomal rearrangements involving the SYT gene located in the

breakpoint region of chromosome 18q11.2 are common among synovial sarcoma

soft tissue tumors. The t(18q11.2) translocation can be identified, for example,

using probes with different labels: the first probe includes FPC nucleic acid

molecules generated from a target nucleic acid sequence that extends distally from

the SYT gene, and the second probe includes FPC nucleic acid generated from a

target nucleic acid sequence that extends 3' or proximal to the SYT gene. When

probes corresponding to these target nucleic acid sequences (e.g., genomic target

nucleic acid sequences) are used in an in situ hybridization procedure, normal

cells, which lack a t(18q11.2) in the SYT gene region, exhibit two fusions

(generated by the two labels in close proximity) signals, reflecting the two intact

copies of SYT. Abnormal cells with a t(18q11.2) exhibit a single fusion signal. In other examples, a target protein produced from a nucleic acid sequence

(e.g., genomic target nucleic acid sequence) is selected that is a tumor suppressor gene that is deleted (lost) in malignant cells. For example, the p16 region

(including D9S1749, D9S1747, p16(NK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p2l is deleted in certain bladder cancers.

Chromosomal deletions involving the distal region of the short arm of chromosome

1 (that encompasses, for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), and the pericentromeric region (e.g., 19p13-19ql3) of chromosome 19 (that encompasses, for example, MAN2B1, ZNF443, ZNF44,

CRX, GLTSCR2, and GLTSCR1) are characteristic molecular features of certain

types of solid tumors of the central nervous system.

The aforementioned examples are provided solely for purpose of

illustration and are not intended to be limiting. Numerous other cytogenetic

abnormalities that correlate with neoplastic transformation and/or growth are

known to those of ordinary skill in the art. Target proteins that are produced by

nucleic acid sequences (e.g., genomic target nucleic acid sequences), which have

been correlated with neoplastic transformation and which are useful in the

disclosed methods, also include the EGFR gene (7p2; e.g., GENBANK TM Accession No. NC-000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK TM Accession No. NC-000008, nucleotides 128817498-128822856), D5S271 (5pl5.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK TM Accession No. NC-000008, nucleotides 19841058 19869049), RB1 (13q14; e.g., GENBANK TM Accession No. NC-000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g., GENBANK TM Accession No. NC-000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANK TM Accession No. NC-000002, complement, nucleotides

151835231-151854620), CHOP (12q13; e.g., GENBANK TM Accession No. NC 000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK T M Accession No. NC-000016, nucleotides 31098954-31110601), FKHR (13pl4; e.g., GENBANK T M Accession No. NC-000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; e.g., GENBANK T M Accession No. NC-000002, complement, nucleotides 29269144 29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK TM Accession No. NC-000011, nucleotides 69165054.69178423), BCL2 (18q21.3; e.g., GENBANK T M Accession No. NC-000018, complement, nucleotides 58941559-

59137593), BCL6 (3q27; e.g., GENBANK T M Accession No. NC-000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (lp 3 2 -p 3 l; e.g., GENBANK T M Accession No. NC-000001, complement, nucleotides 59019051 59022373), TOP2A (17q21-q22; e.g., GENBANK TM Accession No. NC-000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK T M Accession No. NC-000021, complement, nucleotides 41758351 41801948), ERG (21q22.3; e.g., GENBANK T M Accession No. NC-000021, complement, nucleotides 38675671-38955488); ETV1 (7 p 2 l. 3 ; e.g., GENBANK T M Accession No. NC-000007, complement, nucleotides 13897379 13995289), EWS (22q12.2; e.g., GENBANK T M Accession No. NC-000022, nucleotides 27994271-28026505); FL1l (11q24.1-q24.3; e.g., GENBANK TM Accession No. NC-000011, nucleotides 128069199-128187521), PAX3 (2q35 q37; e.g., GENBANK T M Accession No. NC-000002, complement, nucleotides

222772851-222871944), PAX7 (lp36.2-p36.12; e.g., GENBANK TM Accession No. NC-000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK T M Accession No. NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., GENBANK TM Accession No. NC-000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK T M Accession No. NC-000001, complement, nucleotides 40133685 40140274), REL (2pl13-pl2; e.g., GENBANK TM Accession No. NC-000002, TM nucleotides 60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK Accession No. NC-000005, complement, nucleotides 149413051-149473128). In other examples, a target protein is selected from a virus or other

microorganism associated with a disease or condition. Detection of the virus- or

microorganism-derived target nucleic acid sequence (e.g., genomic target nucleic

acid sequence) in a cell or tissue sample is indicative of the presence of the

organism. For example, the target peptide, polypeptide or protein can be selected

from the genome of an oncogenic or pathogenic virus, a bacterium or an

intracellular parasite (such as Plasmodium falciparum and other Plasmodium

species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and

Giardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesia species).

In some examples, the target protein is produced from a nucleic acid

sequence (e.g., genomic target nucleic acid sequence) from a viral genome.

Exemplary viruses and corresponding genomic sequences (GENBANKTM RefSeq

Accession No. in parentheses) include human adenovirus A (NC-001460), human

adenovirus B (NC-004001), human adenovirus C(NC-001405), human adenovirus D (NC-002067), human adenovirus E (NC-003266), human adenovirus F (NC-001454), human astrovirus (NC-001943), human BK polyomavirus (V01109; GI:60851) human bocavirus (NC-007455), human coronavirus 229E (NC-002645), human coronavirus HKU1 (NC-006577), human coronavirus NL63 (NC-005831), human coronavirus OC43 (NC

005147), human enterovirus A (NC-001612), human enterovirus B (NC

001472), human enterovirus C(NC-001428), human enterovirus D (NC

001430), human erythrovirus V9 (NC-004295), human foamy virus (NC 001736), human herpesvirus 1 (Herpes simplex virus type 1) (NC-001806), human herpesvirus 2 (Herpes simplex virus type 2) (NC-001798), human

herpesvirus 3 (Varicella zoster virus) (NC-001348), human herpesvirus 4 type 1

(Epstein-Barr virus type 1) (NC-007605), human herpesvirus 4 type 2 (Epstein

Barr virus type 2) (NC-009334), human herpesvirus 5 strain AD 169 (NC 001347), human herpesvirus 5 strain Merlin Strain (NC-006273), human herpesvirus 6A (NC-001664), human herpesvirus 6B (NC-000898), human herpesvirus 7 (NC-001716), human herpesvirus 8 type M (NC-003409), human herpesvirus 8 type P (NC-009333), human immunodeficiency virus 1 (NC

001802), human immunodeficiency virus 2 (NC-001722), human metapneumovirus (NC-004148), human papillomavirus-1 (NC-001356), human papillomavirus-18 (NC-001357), human papillomavirus-2 (NC-001352), human papillomavirus-54 (NC-001676), human papillomavirus-61 (NC 001694), human papillomavirus-cand90 (NC-004104), human papillomavirus RTRX7 (NC-004761), human papillomavirus type 10 (NC-001576), human papillomavirus type 101 (NC-008189), human papillomavirus type 103 (NC 008188), human papillomavirus type 107 (NC-009239), human papillomavirus type 16 (NC-001526), human papillomavirus type 24 (NC-001683), human papillomavirus type 26 (NC-001583), human papillomavirus type 32 (NC 001586), human papillomavirus type 34 (NC-001587), human papillomavirus type 4 (NC-001457), human papillomavirus type 41 (NC-001354), human papillomavirus type 48 (NC-001690), human papillomavirus type 49 (NC-

001591), human papillomavirus type 5 (NC-001531), human papillomavirus type 50 (NC-001691), human papillomavirus type 53 (NC-001593), human papillomavirus type 60 (NC-001693), human papillomavirus type 63 (NC 001458), human papillomavirus type 6b (NC-001355), human papillomavirus type 7 (NC-001595), human papillomavirus type 71 (NC-002644), human papillomavirus type 9 (NC-001596), human papillomavirus type 92 (NC 004500), human papillomavirus type 96 (NC-005134), human parainfluenza virus 1 (NC-003461), human parainfluenza virus 2 (NC-003443), human parainfluenza virus 3 (NC-001796), human parechovirus (NC-001897), human parvovirus 4 (NC-007018), human parvovirus B19 (NC-000883), human respiratory syncytial virus (NC-001781), human rhinovirus A (NC-001617), human rhinovirus B (NC-001490), human spumaretrovirus (NC-001795), human T-lymphotropic virus 1 (NC-001436), human T-lymphotropic virus 2 (NC-001488). In certain examples, the target protein is produced from a nucleic acid

sequence (e.g., genomic target nucleic acid sequence) from an oncogenic virus,

such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g.,

HPV16, HPV18). In other examples, the target protein produced from a nucleic

acid sequence (e.g., genomic target nucleic acid sequence) is from a pathogenic

virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C

Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a

Cytomegalovirus (CMV), or a Herpes Simplex Virus (HSV).

Examples Example 1 - Synthesis of [4-({[3-({2-[3-(2,5-dioxo-2,5-dihydro-1H pyrrol-1-yl)propanamido]ethylIcarbamoyl)quinoxalin-2-yl]oxyImethyl)-3,5 dimethoxyphenoxy]phosphonic acid (see Scheme 1).

A 2-hydroxyquinoxaline (HQ) hapten was modified with a caging group

that could be released by alkaline phosphatase (AP), namely a caging group

comprising a phosphatase enzyme substrate portion. Secondary anti-species

antibodies (goat-anti-rabbit and goat-anti-mouse) were individually labeled with

either the caged hapten (cHQ) or AP. Thus, any pair of rabbit and mouse primary

antibodies could be tested to determine their proximity to each other. The caged

HQ was synthesized according to the methodology set forth below and as illustrated in Scheme 1, which illustrates the preparation of caged HQ with a maleimide (MAL) reactive group for conjugation to antibodies (cHQ-MAL). 0 0 0 OO OH _oa 0 P,_ O CI 1)NaBH4,THF I 0 ' TEA, EtOAc O 0 2)00 SOCl2. O O' O--0 HO HO CI 3 0 1 2

0 0 4 0 TEADM HO-N5 0 -OH HO-M-OH 0 O

o H 00 O No

0 NH2 TEA,DM -- ' N-N0O 7 0 8H

Compound 2. A soln. of 2,6-dimethoxy-4-hydroxybenzaldehyde (5.00 g, 27.4 mmol) and triethylamine (4.17 g, 5.74 mL, 41.2 mmol) in EtOAc (25 mL) was cooled to0 O°C in an ice bath in stirring. Diethyl chlorophosphate (4.73 g, 27.4

mmol) was then added dropwise over a period of 5 min. The reaction was allowed

to warm to rt, followed by stirring at room temperature ("rt") for about 6 h (check

HPLC to confirm reaction was greater than about 90% complete). The reaction

mixture was quenched by addition of 1M HCl (100 mL) and the organic layer was

separated and collected. An additional quantity of EtOAc (100 mL) was added and

the organics were extracted with 1M HCl (2 x 100 mL), sat'd NaHCO 3 (3 x 100

mL) and brine (100 mL). The organic layer was dried over MgSO 4 and the solvents

removed under reduced pressure to give compound 2 as a light brown viscous oil

(7.95 g, 91% yield). Compound 3. A soln. of compound 2 (7.95 g, 25.0 mmol) in THF (about 100 mL) was cooled to about 0°C in an ice bath with stirring. NaBH 4 (1.42 g, 37.5

mmol) was crushed to fine powder with a mortar and pestle and was added portion-wise, followed by stirring at rt for about 4 h (check HPLC to confirm reaction completion). The reaction mixture was carefully quenched by addition of

1M HCl until bubbling ceased. The majority of the THF was then removed under

reduced pressure. The resulting reaction mixture was extracted with EtOAc (3 x

100 mL). The organic layers were collected and combined, followed by washing

with brine (about 100 mL) and drying over MgSO 4 . The solvents were then

removed under reduced pressure. The resulting light brown viscous oil was

dissolved in dry CH 2Cl 2 (about 50 mL) followed by cooling to about 0 °C in an ice

bath under an atmosphere of N 2 with stirring. SOCl2 (4.46 g, 2.72 mL, 37.5 mmol)

was then added dropwise over a period of about 5 min. The reaction mixture was

allowed to warm to rt, followed by stirring at rt for an additional 1 h (check HPLC

to confirm reaction completion). The reaction mixture was carefully quenched by

addition of sat'd NaHCO 3 until bubbling ceased. The resulting reaction mixture

was extracted with CH 2Cl2 (3 x 100 mL). The organic layers were collected and

combined, followed by washing with brine (100 mL) and drying over MgSO 4 . The solvents were then removed under reduced pressure to give compound 3 as an off

white low melting solid (7.54 g, 89% yield). Compound 5. 3-Hydroxy-2-quinoxalinecarboxylic acid (3.00 g, 15.8 mmol) was dissolved in DMF (50 mL), followed by addition of 4-dimethylaminopyridine (2.12 g, 17.4 mmol). The reaction mixture was stirred at rt until the 4

dimethylaminopyridine dissolved, at which point N,N'-disuccinimidyl carbonate

(4.45 g, 17.4 mmol) was added. The reaction mixture was stirred at rt for 30 min

(Check HPLC to confirm reaction completion. If the reaction was not complete,

additional 0.1 eq portions of DSC were added until HPLC showed complete

conversion). A soln. of N-Boc-ethylenediamine (3.04 g, 19.0 mmol) and

triethylamine (2.40 g, 3.30 mL, 23.7 mmol) in DMF (10 mL) was then added, and the reaction was stirred at rt for 1 h (check HPLC for reaction completion). The

reaction mixture was then poured onto a vigorously stirring soln. of 1M HCl (250

mL). The resulting precipitate was collected by vacuum filtration, washed several

times with water, and dried under high-vacuum, giving compound 5 as a yellow

solid (5.10 g, 97% yield). Compound 6. Compound 5 (2.50 g, 7.52 mmol) was dissolved in DMF (50 mL), followed by addition of compound 3 (3.82 g, 11.3 mmol) and K2CO3 (5.20 g,

37.6 mmol). The reaction vessel was sealed and heated to 55 °C in an oil bath with

vigorous stirring for 2 h (Check HPLC to confirm reaction completion. If the

reaction was not complete, additional 0.1 eq portions of compound 3 were added

until HPLC showed complete consumption of compound 5). The reaction mixture

was filtered and the filtrate carefully quenched by addition of 1M HCl until

bubbling ceased. The resulting reaction mixture was extracted with EtOAc (3 x

100 mL). The organic layers were collected and combined, followed by washing

with brine (100 mL) and drying over MgSO 4 . The solvents were then removed

under reduced pressure to give a light yellow viscous oil. The resulting residue was

purified by flash chromatography (Biotage Snap 50; hex:EA 1:0 to 1:4) to give compound 6 as a light yellow solid (3.15 g, 66% yield). Compound 7. Compound 6 (3.15 g, 4.96 mmol) was dissolved in dry CH 2Cl2 (25 mL) followed by dropwise addition of trimethylsilyl bromide (3.80 g, 3.28 mL, 24.8 mmol) over a period of 5 min. The vessel was sealed and the

reaction mixture was stirred at rt for 16 h (check HPLC to confirm reaction

completion). MeOH (25 mL) was then added and the solvents were removed under

reduced pressure. The resulting solid was triturated with CH 2Cl 2 (100 mL) and the

resulting solid was collected by vacuum filtration, giving compound 7 as a light

yellow solid (2.06, 87% yield). Compound 8. Compound 7 (2.06 g, 4.31 mmol) was suspended in DMF (10 mL), followed by addition of triethylamine (654 mg, 900 pL, 6.46 mmol) and finally 3-maleimidopropionic acid NHS ester (1.15 g, 4.31 mmol). The reaction vessel was sealed and the reaction mixture was vigorously stirred at room

temperature for about 4 h (check HPLC to confirm reaction completion). The

reaction mixture was then diluted with MeOH (10 mL) and directly purified by

prep RP-HPLC (0.05% TFA in H 20:MeCN 99:1 to 5:95 over 40 min) in 5 portions to give compound 8 as a light yellow solid (1.95 g, 72% yield). The maleimide functionalized HQ (cHQ-MAL) was conjugated to both

goat-anti-rabbit and goat-anti-mouse IgG antibodies by two methodologies, via

disulfide or lysine groups. In the disulfide method, the IgG was reduced by

dithiothreitol (DTT) and then treated with an excess of cHQ-MAL. For the lysine

method, the IgG was treated with an excess of Traut's reagent (2-iminothiolane

hydrochloride) before adding an excess of cHQ-MAL. In both cases the conjugates were purified by size exclusion chromatography (SEC) and characterized by gel electrophoresis and UV-Vis spectroscopy.

Example 2-Confirmation of blocking of caging group

To confirm that the caging group of the caged hapten-antibody conjugate

from Example 1 blocked an anti-hapten antibody from binding to the respective

native (i.e. unmasked) hapten, the cHQ conjugate was tested with single IHC

detection. FIG. 5 illustrates an IHC staining scheme where a single antigen (114) was detected with a secondary antibody conjugated to cHQ, i.e. a cHQ caged hapten-antibody conjugate (103A). Following introduction of the cHQ caged hapten-antibody conjugate (103A), an AP enzyme (i.e. free, unconjugated AP) (113) was introduced to unmask the haptenofthecagedhapten-antibody conjugate. The resulting unmasked hapten (103B) was able to be recognized by an anti-hapten antibody (115). In some embodiments, the anti-hapten antibody (115) is conjugated to one or more enzymes (FIG. 5 illustrates the conjugation to 3 HRP enzymes). With reference to FIGS. 6A - 6C, mouse-anti-PSA bound to its target epitope on prostate tissue and was subsequently bound by goat-anti-mouse conjugated to cHQ (GAM-cHQ). Control slides (e.g. FIG. 6C) were generated using the goat-anti-mouse conjugated to uncaged HQ (GAM-HQ). Serial sections were exposed to two different conditions, namely (i) solutions with free (unconjugated) AP, pH adjust buffer and AP cofactors (magnesium salts); and (ii) solutions without AP, but with pH adjust buffer and AP cofactors (magnesium salts). FIG. 6A illustrates tissue treated with solution comprising AP; while FIG. 6B illustrates untreated tissue, i.e. solution without AP. FIG. 6C illustrates a control slide with a secondary antibody labeled with uncaged HQ. The sample tissues that were treated with AP under conditions favorable for the enzyme had the opportunity to allow unmasking of the caged hapten to occur (FIG. 6A). The negative sample tissues (no AP) did not have the opportunity for controlled unmasking to occur (FIG. 6B). These figures clearly illustrate that the slide that was treated with a GAM-cHQ but was not exposed to AP shows no positive staining (FIG. 6B). However, the slide that had both the GAM-cHQ and AP shows positive staining (FIG. 6A) and matches the non-caged control slide (FIG. 6C).

This tested both the effectiveness of the caging group and its stability to the assay

conditions.

Example 3-Measurement of Protein Proximity

A number of known positive and putative negative systems were

investigated to test the ability of the disclosed conjugates to enable measurement of

protein proximity. E-cadherin and beta-catenin were chosen as known positive

systems since the biology dictates that these proteins are in close contact to each

other in a normal cell. Her2/beta-catenin and EGFR/beta-catenin were chosen as

pairs of protein that are in the same cell compartment (co-localized) but not

expected to interact with each other (not proximal) and therefore should not

generate signal in any proximity assay. The antibody pairs were also chosen so

that there was one rabbit and one mouse primary antibody to allow the use of anti

species secondary detections.

With reference to the schemes outlined in FIGs. 2, 4, and 12, following

introduction of the primary antibodies (e.g. an antibody conjugated to a hapten, a

caged hapten-antibody conjugate, and an unmasking enzyme-antibody conjugate)

and secondary antibodies (e.g. an anti-hapten antibody conjugated to an enzyme),

and subsequent unmasking of the caged hapten, the uncaged HQ was detected with

a mouse-anti-HQ conjugated to horseradish peroxidase (HRP). At this point either

DAB (3,3'-diaminobenzidine), silver or a tyramide-chromogen could be used to

visualize the proximity signal. A HRP/tyramide-hapten amplification step can also

be included to boost the intensity (sensitivity) of the system (e.g. tyramide-HQ

based optiView Amplification kit). A general procedure for the automated caged hapten proximity assay was

used to test FFPE cases: The slides were deparaffinized using a deparaffinizing

solution of DISCOVERY EZ Prep (Ventana, p/n 950-100) or DISCOVERY Wash (Ventana, p/n 950-510). Heat induced epitope retrieval was performed on the slide

using DISCOVERY CCl (Ventana, p/n 950-124) at 95 °C for a time dependent on the identity of the two primary antibodies (0-92 minutes). DISCOVERY Inhibitor (Ventana, p/n 760-4840) was then applied to the slide to quench any endogenous

peroxidase in the sample. A rabbit anti-target 1 antibody was co-incubated with a

mouse anti-target 2 antibody at 37 °C for 16-32 minutes (dependent on the

identities of the primary antibodies). Then a 30 pg/mL goat anti-mouse alkaline phosphatase conjugate was applied to the slide and incubated for 12 minutes. The slide was rinsed and a 20 pg/mL solution of the caged hapten goat anti-rabbit conjugate was applied to the slide for 16 minutes. After the incubation, a 500 mM tris buffer solution pH 10.0 and a 490 mM MgCl 2 solution were applied to the slide to enable the de-caging of the caged hapten. After the de-caging of the HQ, a mouse-anti-HQ HRP (Ventana, p/n 760-4820) conjugate was applied to the slide to bind the uncaged HQ. In some cases the Amp HQ tyramide amplification kit

(Ventana, p/n 760-052) was used to help boost the signal intensity. Proximal

proteins were visualized using the Chromomap DAB kit (Ventana, p/n 760-159),

followed by counterstaining with Hematoxylin II (Ventana, p/n 790-2208) and

Bluing Reagent (Ventana, p/n 760-2037). FIGs. 7A - 7C illustrate the results of this experiment and illustrate

examples of proteins with positive proximity. FIG. 7A illustrates a single IHC

DAB stain for Beta-Catenin (e.g. utilizing detection techniques comprising an

antibody specific for Beta-Catenin, an anti-species antibody conjugated to an

enzyme, and DAB). FIG. 7B illustrates a caged hapten proximity signal for Beta

Catenin and E-Cadherin (utilizing a caged hapten-antibody conjugate acted upon

by an unmasking enzyme-antibody conjugate). FIG. 7C illustrates a single IHC

DAB stain for E-Cadherin (e.g. utilizing detection techniques comprising an

antibody specific for E-Cadherin, an anti-species antibody conjugated to an

enzyme, and DAB).

FIGs 8A - 8C illustrates the results of IHC DAB staining and illustrate

examples of co-localized proteins without proximity as detected with the

conjugates and methods described herein. FIG. 8A illustrates a single IHC DAB

stain for EGFR; FIG. 8B illustrates the absence of a caged hapten proximity signal

for EGFR and E-Cadherin; and FIG. 8C illustrates a single IHC DAB stain for E Cadherin.

In addition, FIGs. 9A and 9B illustrate examples of co-localized proteins

with and without proximity on a different tissue type. Here, FIG. 9A illustrates

positive caged hapten proximity signal for Beta-catenin and E-Cadherin, while

FIG. 9B illustrates the lack of a caged hapten proximity signal for Beta-catenin and

Her2. These examples illustrate that the assay is applicable to different tissue

types, but generates the same type of results.

The signal intensity output from the above-described assays may be

"dialed-in" a number of ways. The use of on-market signal amplification is easily

applied to the presently disclosed technology and is believed to easily boost the

signal (see, e.g. the amplification techniques described in U.S. Publication No.

2012/0171668, and PCT/EP2015/0533556, the disclosures of which are hereby incorporated by reference herein).

As illustrated in FIGS. 10A and 10B, the number of caged haptens

conjugated to the secondary antibody also allows the sensitivity of the assay to be

adjusted to fit a given system. In this example, FIG. 10A illustrates a sample

labeled with goat-anti-rabbit and 4 (four) cHQ labels while FIG. 10B illustrates a

sample labeled with goat-anti-rabbit and 9 (nine) cHQ labels. Here, the

comparative increase in signal intensity is based on the increased number of caged

haptens conjugated to the secondary antibody, where FIG. 10B clearly shows the

increase in staining intensity as compared with FIG. 10A.

Example 4-Detection with Different HRP Systems

Different HRP systems providing different detectable signals (e.g. colors)

were tested. For example, FIGs. 11A and 11B illustrate detection with silver

(11A) and Tyramide-TAMRA (11B). Both systems were able to detect proximal

protein targets in a sample.

Example 5-Multiplex detection of Total Protein and Proximal Protein

Signal

FIG. 12 is a schematic illustrating the multiplex detection of one total

protein (Target 2, AP-Yellow) and the proximal protein signal (Target 1 + Target

2, HRP-Silver). Any type of HRP detection system may be used to show the

proximity signal and any AP detection system (e.g. naphthol phosphate/Fast Red,

BCIP/NBT, quinone methide (QM)-chromogen) may be used to visualize the total

protein.

The detection of proximal proteins utilizing HRP-DAB (FIG. 13A) and HRP-Silver with the addition of a total protein stain for one of the targets has also

been demonstrated (FIG. 13B). These experiments were performed with E

cadherin and Beta-catenin on tonsil tissue. In addition, FIGS. 14A and 14B

illustrate the use of different HRP detection systems to visualize the proximity signal along with total protein, utilizing an E-cadherin and Beta-catenin duel stain.

FIG. 14A illustrates proximity detected with HRP-Purple and Beta-Catenin

detected with AP-Yellow, while FIG. 13B illustrates proximity detected with HRP

Silver and Beta-Catenin detected with AP-Yellow.

Example 6-PD1/PD-L1 Binding of the ligand PD-L1 to its receptor PD-1 modulates the activation

or inhibition of T-cells. Tumor cells can use this interaction to evade the immune

system. Detection of a PD-Li/PD-1 complex may provide a better indication of

the blockade status of the immune checkpoint within a tumor than simple

measurement of either protein by itself.

FIG. 16 illustrates a caged hapten proximity signal for PD-1 and PD-L1

(utilizing a caged hapten-antibody conjugate acted upon by an unmasking enzyme

antibody conjugate) in a NSCLC. The purple signal represents the detection of PD

Li/PD-1 complexes.

The caged hapten proximity assay was used to test FFPE cases of NSCLC

and tonsil. The cases were run on the DISCOVERY ULTRA instrument, fully

automated. The slides were deparaffinized using a deparaffinizing solution of

DISCOVERY EZ Prep (Ventana, p/n 950-100) or DISCOVERY Wash (Ventana, p/n 950-510). Heat induced epitope retrieval was performed on the slide using

DISCOVERY CCi (Ventana, p/n 950-124) at 95 °C for 64 minutes. DISCOVERY Inhibitor (Ventana, p/n 760-4840) was then applied to the slide to quench any endogenous peroxidase in the sample. Rabbit anti-PD-Li (SP263)

(Ventana, p/n 790-4905) was co-incubated with a mouse anti-PD-1 (NAT105)

(Ventana, p/n 760-4895) for 32 minutes at 37 °C. For a negative control the mouse

anti-PD-1 was omitted. For the remainder of the run, the slide was incubated at 37

°C. A 30 pg/mL goat anti-mouse alkaline phosphatase conjugate was applied to

the slide and incubated for 12 minutes. The slide was rinsed and a 20 pg/mL

solution of the caged hapten goat anti-rabbit conjugate was applied to the slide for

16 minutes. After the incubation, a 500 mM tris buffer solution pH 10.0 and a 490

mM MgCl2 solution were applied to the slide to enable the de-caging of the caged

hapten. After the de-caging of the HQ, a mouse-anti-HQ HRP (Ventana, p/n 760

4820) conjugate was applied to the slide to bind the uncaged HQ. Proximal

proteins were visualized using the DISCOVERY Purple kit (Ventana, p/n 760-

229). The Amp HQ tyramide amplification kit (Ventana, p/n 760-052) was used to help boost the signal intensity when necessary.

FIG. 17 illustrates a caged hapten proximity signal for PD-L1 and PD-1

along with the total protein detection for PD-1. After detection of proximal

proteins with DISCOVERY Purple then the uncaging enzyme (alkaline

phosphatase) was detected with DISCOVERY Yellow (Ventana, p/n 760-239) to

visualize the PD-1 on the tissue.

FIG. 18 illustrates a negative control for a caged hapten proximity signal

for PD-L1 and PD-1 in tonsil tissue. The primary antibody for PD-1 was omitted

from the assay. No signal was observed demonstrating the specificity of the

detection reagents for the protein complex.

In all cases the slides were counterstained with Hematoxylin II (Ventana,

p/n 790-2208) and Bluing Reagent (Ventana, p/n 760-2037). Example 7-PDi/PD-L1 combined with CD8 FIG. 19 illustrates an example of combining the caged hapten proximity

signal for PD-L1 and PD-1 with the subsequent detection of another biomarker in

tonsil tissue. In this case CD8 was detected after the PD-L/PD1 complex to

generate an assay that visualized the CD8 cells that were involved in PD-L/PD-1

interactions. For multiplexing, the PD-1 and PD-L1 proximity was first detected as

previously described. The slides were heated to 90 °C for 8 minutes to assist in the

elution of the previously bound primaries. Rabbit anti-CD8 (SP57) (Ventana, p/n

790-4460) was applied to the slide and incubated for 16 minutes at 37 °C. The

anti-CD8 was detected using UltraMap Rb AP (Ventana, p/n 760-4314) followed

by the DISCOVERY Yellow chromogen. The slides were counterstained with

Hematoxylin II (Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n 760

2037). Example 8-Her2/Her3 The caged hapten proximity assay was used to test FFPE cases of BT-474

cell lines. The cases were run on the DISCOVERY ULTRA instrument, fully

automated. The slides were deparaffinized using a deparaffinizing solution of

DISCOVERY CCi (Ventana, p/n 950-124) at 95 °C for 64 minutes.

DISCOVERY Inhibitor (Ventana, p/n 760-4840) was then applied to the slide to quench any endogenous peroxidase in the sample. Rabbit anti-Her2 (4B5) (Ventana, p/n 790-2991) was co-incubated with a mouse anti-Her3 (SPM738) (Spring, p/n E19260) for 32 minutes at 37 °C. Then a 30 pg/mL goat anti-mouse alkaline phosphatase conjugate was applied to the slide and incubated for 12 minutes. The slide was rinsed and a 20 pg/mL solution of the caged hapten goat anti-rabbit conjugate was applied to the slide for 16 minutes. After the incubation, a 500 mM tris buffer solution pH 10.0 and a 490 mM MgCl 2 solution were applied to the slide to enable the de-caging of the caged hapten. After the de-caging of the HQ, a mouse-anti-HQ HRP (Ventana, p/n 760-4820) conjugate was applied to the slide to bind the uncaged HQ. The Amp HQ tyramide amplification kit (Ventana, p/n 760-052) was used to help boost the signal intensity. Proximal proteins were visualized using the Chromomap DAB kit (Ventana, p/n 760-159), followed by counterstaining with Hematoxylin II (Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n 760-2037). FIGs. 20A - 20C illustrate the results of this experiment and illustrate examples of proteins with positive proximity. FIG. 20A illustrates a single IHC DAB stain for Her2 (e.g. utilizing detection techniques comprising an antibody specific for Her2, an anti-species antibody conjugated to an enzyme, and DAB). FIG. 20B illustrates a caged hapten proximity signal for Her2 and Her3 (utilizing a caged hapten-antibody conjugate acted upon by an unmasking enzyme-antibody conjugate). FIG. 20C illustrates a single IHC DAB stain for Her3 (e.g. utilizing detection techniques comprising an antibody specific for Her3, an anti-species antibody conjugated to an enzyme, and DAB). Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. For example, an "antibody" used in accordance with the present invention may be a whole antibody or a fragment of an antibody that is effective in binding to a desired target site. Also, when appropriate, an "antibody" of the present invention may be substituted with a targeting moiety (e.g., ligand peptide, small molecule, etc.). For example, if the tumor cell or the immune cell has a specific, differentiating and unique cell surface receptor, then a corresponding targeting moiety may be used in accordance with the present invention to differentiate tumor cells from immune cells.

Claims

Divisional of AU 2021218128 P296830D2 (R74327PCAUT2) 71 Claims

1. A method for analyzing a sample to determine whether a first target is proximal to a second target, the method comprising:

(a) forming a second target-unmasking enzyme-antibody conjugate complex;

(b) forming a first target-caged hapten-antibody conjugate complex;

(c) unmasking the caged hapten of the first target-caged hapten-antibody conjugate complex to form a first target-unmasked hapten-antibody conjugate complex;

(d) contacting the sample with first detection reagents to label the first target unmasked hapten-antibody conjugate complex or the first target; and

(e) detecting the labeled first target-unmasked hapten-antibody conjugate complex or labeled first target.

2. The method of claim 1, wherein the first detection reagents comprise (i) a secondary antibody specific to the unmasked hapten of the first target-unmasked hapten-antibody complex, the secondary antibody conjugated to a first enzyme such that the secondary antibody labels the first target-unmasked hapten-antibody complex with the first enzyme; and (ii) a first substrate for the first enzyme.

3. The method of claim 2, wherein the first substrate is a chromogenic substrate or a fluorescent substrate.

4. The method of claim 1, wherein the first detection reagents include amplification components to label the unmasked enzyme of thefirst target-unmasked hapten-antibody conjugate complex with a plurality of first reporter moieties.

5. The method of claim 4, wherein the plurality of first reporter moieties are haptens.

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6. The method of claim 5, wherein thefirst detection reagents further comprise secondary antibodies specific to the plurality of first reporter moieties, each secondary antibody conjugated to a second reporter moiety.

7. The method of claim 5, wherein the second reporter moiety is selected from the group consisting of an amplification enzyme or a fluorophore.

8. The method of claim 7, wherein the second reporter moiety is an amplification enzyme and wherein the first detection reagents further comprise a first chromogenic substrate or a fluorescent substrate for the amplification enzyme.

9. The method of claim 1, further comprising contacting the sample with a second substrate specific for the unmasking enzyme of the second target-unmasking enzyme-antibody conjugate complex and detecting signals corresponding to a product of a reaction between the second substrate and the unmasking enzyme.

10. A method for analyzing a sample to determine whether a first target is proximal to a second target, the method comprising:

(a) forming a second target-unmasking enzyme-antibody conjugate complex;

(b) forming a first target-caged hapten-antibody conjugate complex;

(d) performing a signal amplification step to label thefirst target-unmasked hapten antibody conjugate complex with a plurality of reporter moieties; and

(e) detecting the plurality of reporter moieties.

11. The method of claim 10, wherein the plurality of reporter moieties are haptens; and wherein the method further comprises introducing secondary antibodies specific to the plurality of first reporter moieties, each secondary antibody conjugated to a second reporter moiety.

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12. The method of claim 11, wherein the second reporter moiety is an amplification enzyme and wherein the method further comprises introducing a chromogenic substrate or a fluorescent substrate for the amplification enzyme.

13. The method of claim 10, further comprising detecting a total amount of target in the sample.

14. A method for analyzing a sample to determine whether a first target is proximal to a second target, the method comprising:

(a) forming a second target-unmasking enzyme-antibody conjugate complex;

(b) forming a first target-caged hapten-antibody conjugate complex;

(c) performing a decaging step such that an unmasking enzyme of the second target-unmasking enzyme-antibody conjugate complex reacts with an enzyme substrate portion of the first target-caged hapten-antibody conjugate complex to form a first target-unmasked hapten-antibody conjugate complex;

15. The method of claim 14, wherein the decaging step comprises changing the temperature of the sample.

16. The method of claim 14, wherein the decaging step comprises altering a pH of the sample.

17. The method of claim 14, wherein the decaging step comprises introducing one or more washing steps.

18. The method of claim 14, wherein the decaging step comprises adding cofactors for the unmasking enzyme.

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19. The method of claim 14, further comprising detecting a total amount of target in the sample.

20. A caged hapten having Formula (IA):

Y X Hapten- -A Leaving Group-Enzyme Substrate (IA),

wherein

Y is selected from a carbonyl-reactive group, an amine-reactive group, or a thiol-reactive

group;

X is a bond, or a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 30 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of 0, N, or S;

A is a bond, or a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of 0, N, or S;

q is 0 or 1;

'Enzyme Substrate' is a phosphate group, an ester group, a sulfate group, a glycoside group, an amide group, a urea group, or a nitro group; and

'Leaving Group' is a 5-, 6-, or 7-membered aromatic ring or heterocyclic ring, optionally substituted with a halogen, a -S-alkyl group having between 1 and 4 carbon atoms; an -0 alkyl group having between 1 and 4 carbon atoms; a -N(H)-alkyl group having between 1 and 4 carbon atoms; a -N-(alkyl)2 group having between 1 and 6 carbon atoms; or a branched or unbranched, substituted or unsubstituted alkyl group having between 1 and 4 carbon atoms.

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21. A caged hapten having Formula (IB):

Y X Hapten- A Enzyme Substrate (IB),

wherein

group;

A is a bond, or a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of O, N, or S;

qis0or 1; and

'Enzyme Substrate' is a phosphate group, an ester group, a sulfate group, a glycoside group, an amide group, a urea group, or a nitro group.